U.S. patent application number 14/906643 was filed with the patent office on 2016-06-23 for fluidic cartridge and method for processing a liquid sample.
The applicant listed for this patent is ATLAS GENETICS LIMITED. Invention is credited to Ben Arlett, Jay Kendall Taylor.
Application Number | 20160175835 14/906643 |
Document ID | / |
Family ID | 49167103 |
Filed Date | 2016-06-23 |
United States Patent
Application |
20160175835 |
Kind Code |
A1 |
Taylor; Jay Kendall ; et
al. |
June 23, 2016 |
FLUIDIC CARTRIDGE AND METHOD FOR PROCESSING A LIQUID SAMPLE
Abstract
A fluidic cartridge for processing a liquid sample comprises a
main channel for passing the liquid sample therethrough from an
upstream end to a downstream end; and one or more branch channels
that join the main channel for introducing liquid and gas into the
main channel after the liquid sample has passed downstream of the
one or more branch channels. The one or more branch channels
including a first branch channel, wherein the first branch channel
comprises: a gas inlet for introducing a gas into the first branch
channel; a liquid inlet for introducing a liquid into the first
branch channel; and a valve configured to move between a closed
position in which it prevents liquid and gas in the first branch
channel from passing into the main channel and an open position in
which it permits liquid and gas in the first branch channel to pass
into the main channel.
Inventors: |
Taylor; Jay Kendall;
(Ottawa, CA) ; Arlett; Ben; (Bristol, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ATLAS GENETICS LIMITED |
Trowbridge Wiltshire |
|
GB |
|
|
Family ID: |
49167103 |
Appl. No.: |
14/906643 |
Filed: |
July 28, 2014 |
PCT Filed: |
July 28, 2014 |
PCT NO: |
PCT/GB2014/052306 |
371 Date: |
January 21, 2016 |
Current U.S.
Class: |
436/180 ;
422/501; 422/522 |
Current CPC
Class: |
B01L 2400/06 20130101;
G01N 1/38 20130101; B01L 2400/0622 20130101; B01L 2200/16 20130101;
B01L 2200/10 20130101; B01L 2300/0681 20130101; B01L 2200/141
20130101; B01L 2400/0683 20130101; B01L 2300/0816 20130101; B01L
3/502 20130101; B01L 7/52 20130101; B01L 3/567 20130101; B01L
2300/123 20130101; B01L 2200/0684 20130101; B01L 2400/0481
20130101; B01L 2400/0638 20130101; B01L 2300/0867 20130101; B01L
2200/0673 20130101; B01L 3/502738 20130101; B01L 2300/0864
20130101; B01L 2200/027 20130101; B01L 2400/0487 20130101; B01L
2200/0605 20130101; B01L 2400/0655 20130101; B01L 2300/1883
20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; G01N 1/38 20060101 G01N001/38 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 29, 2013 |
GB |
1313515.7 |
Claims
1. A fluidic cartridge for processing a liquid sample, comprising:
a main channel for passing the liquid sample therethrough from an
upstream end to a downstream end; and one or more branch channels
that join the main channel for introducing liquid and gas into the
main channel after the liquid sample has passed downstream of the
one or more branch channels, the one or more branch channels
including a first branch channel, wherein the first branch channel
comprises: a gas inlet for introducing a gas into the first branch
channel; a liquid inlet for introducing a liquid into the first
branch channel; and a valve configured to move between a closed
position in which it prevents liquid and gas in the first branch
channel from passing into the main channel and an open position in
which it permits liquid and gas in the first branch channel to pass
into the main channel.
2. The fluidic cartridge of claim 1, wherein the one or more branch
channels further comprises a second branch channel which joins the
main channel downstream of the first branch channel, wherein the
second branch channel comprises: a gas inlet for introducing a gas
into the second branch channel; a liquid inlet for introducing a
liquid into the second branch channel; and a valve configured to
move between a closed position in which it prevents liquid and gas
in the second branch channel from passing into the main channel and
an open position in which it permits liquid and gas in the second
branch channel to pass into the main channel.
3. The fluidic cartridge of any preceding claim, wherein the or
each gas inlet on the one or more branch channels comprises a gas
inlet valve to prevent liquid and gas flowing from the branch
channel through the gas inlet.
4. The fluidic cartridge of claim 1 or claim 2, wherein the or each
valve in the one or more branch channels is spaced from the main
channel, thereby forming a dead-leg in the branch channel between
the valve and the main channel.
5. The fluidic cartridge of claim 1 or claim 2, wherein the or each
valve in the one or more branch channels is located at a junction
between the branch channel and the main channel.
6. The fluidic cartridge of any preceding claim, wherein the or
each gas inlet is located further from the junction of the branch
channel with the main channel than the or each liquid inlet.
7. The fluidic cartridge of claim 3, wherein the or each gas inlet
valve is located further from the junction of the branch channel
with the main channel than the or each liquid inlet, and the or
each gas inlet is located further from the junction of the branch
channel with the main channel than the or each gas inlet valve.
8. The fluidic cartridge of any preceding claim, wherein the or
each liquid inlet on the one or more branch channels is coupled to
a liquid chamber.
9. The fluidic cartridge of claim 8, wherein the or each liquid
chamber is a collapsible blister adapted, when it is collapsed, to
eject a liquid contained therein through the liquid inlet, into the
branch channel, and into the main channel, for introducing the
liquid into the main channel after the liquid sample has passed
downstream of the one or more branch channels.
10. The fluidic cartridge of claim 8 or 9, wherein the or each
liquid chamber contains a reagent or a buffer such as a lysis
buffer, a wash buffer or an elution buffer.
11. The fluidic cartridge of any one of claims 8 to 10, when
dependent on claim 2 or any claim dependent on claim 2, wherein the
liquid chamber coupled to the liquid inlet in the first branch
channel contains a wash buffer, and wherein the liquid chamber
coupled to the liquid inlet in the second branch channel contains
an elution buffer.
12. The fluidic cartridge of any preceding claim, further
comprising a pneumatic interface for connecting to a source of
positive and/or gauge pressure, the pneumatic interface comprising
a plurality of ports, and wherein the or each valve or gas inlet
valve in the one or more branch channels is a
pneumatically-actuated valve coupled to at least one port in the
pneumatic interface such that it may be actuated by the source of
positive and/or gauge pressure.
13. The fluidic cartridge of claim 12, when dependent on claim 2,
wherein the first and second gas inlet valves in the first and
second branch channels, respectively, are coupled to the same port
in the pneumatic interface, such that the first and second gas
inlet valves may be actuated simultaneously.
14. The fluidic cartridge of any preceding claim, wherein the or
each gas inlet on the one or more branch channels is coupled to the
pneumatic interface for connection to a gas supply.
15. The fluidic cartridge of claim 14, wherein the or each gas
inlet on the one or more branch channels is coupled to the
pneumatic interface for connection to a gas supply for passing a
gas through the gas inlet, into the branch channel, and into the
main channel, for introducing the gas into the main channel after
the liquid sample has passed downstream of the one or more branch
channels.
16. The fluidic cartridge of claim 12, wherein the or each
pneumatically-actuated valve comprises a valve chamber having first
and second openings connected to the branch channel; and a flexible
membrane movable between a closed position, in which the flexible
membrane seals against the first and second openings to prevent
fluid flow through the branch channel, and an open position, in
which the flexible membrane is spaced apart from the first and
second openings to permit fluid to flow through the branch
channel.
17. The fluidic cartridge of claim 16, when dependent on claim 10,
wherein the or each valve further comprises a fluid passageway
having an opening in the valve chamber, the opening separated from
the first and second openings by the flexible membrane, wherein the
fluid passageway is coupled to a port in the pneumatic interface
for applying a positive or gauge pressure in the valve chamber to
move the flexible membrane between the open and closed
positions.
18. The fluidic cartridge of any one of claims 12 to 17, when
dependent on claim 2 or any claim dependent on claim 2, wherein the
pneumatic interface comprises first to third ports, and wherein the
first to third ports are respectively coupled to: i. the valve in
the first branch channel; ii. the valve in the second branch
channel; and iii. the gas inlet valve in the first branch channel
and the gas inlet valve in the second branch channel; such that the
respective valves may be actuated by the source of positive and/or
gauge pressure acting through the respective ports.
19. A method of processing a liquid sample in a fluidic cartridge,
the cartridge comprising a main channel and one or more branch
channels that join the main channel, including a first branch
channel comprising a gas inlet, a liquid inlet and a valve, the
method comprising; a) passing a liquid sample through the main
channel; b) supplying a gas to the gas inlet; c) opening the valve
on the first branch channel and passing a gas from the gas inlet
through the first branch channel and into the main channel to
evacuate any residual liquid sample in the first branch channel; d)
ceasing the supply of gas; e) passing a liquid from the liquid
inlet through the first branch channel and into the main channel;
f) supplying a gas to the gas inlet; and g) passing a gas from the
gas inlet through the first branch channel and into the main
channel to evacuate any residual liquid from the first branch
channel.
20. The method of claim 19, wherein the fluidic cartridge further
comprises a gas inlet valve on the first branch channel, and
wherein steps (b) and (f) of supplying a gas to the gas inlet
further comprise opening the gas inlet valve, and wherein step (d)
of ceasing the supply of gas further comprises closing the gas
inlet valve and before ceasing the supply of gas.
21. A method of processing a liquid sample in a fluidic cartridge,
the cartridge comprising a main channel having an upstream end and
a downstream end and one or more branch channels that join the main
channel, including a first branch channel and second branch channel
downstream of the first branch channel, each branch channel
comprising a gas inlet, a gas inlet valve, a liquid inlet and a
valve, the method comprising: a) passing a liquid sample through
the main channel; b) supplying a gas to the gas inlet on the first
branch channel and the gas inlet on the second branch channel; c)
opening the valves and the gas inlet valves on the first and second
branch channels and passing a gas from the gas inlets on the first
and second branch channels through the first and second branch
channels and into the main channel to evacuate any residual liquid
sample in the first and second branch channels; d) closing the
valve and the gas inlet valve on the second branch channel and the
gas inlet valve on the first branch channel; e) ceasing the supply
of gas to the gas inlet on the first branch channel and the gas
inlet on the second branch channel; f) passing a liquid from the
liquid inlet on the first branch channel through the first branch
channel and into the main channel; g) supplying a gas to the gas
inlet on the first branch channel and the gas inlet on the second
branch channel; h) opening the valve and the gas inlet valve on the
second branch channel and the gas inlet valve on the first branch
channel and passing a gas from the gas inlets on the first and
second branch channels through the first and second branch channels
and into the main channel to evacuate any residual liquid in the
first and second branch channels; i) closing the valve and the gas
inlet valve on the first branch channel and the gas inlet valve on
the second branch channel; j) ceasing the supply of gas to the gas
inlet on the first branch channel and the gas inlet on the second
branch channel; k) passing a liquid from the liquid inlet on the
second branch channel through the second branch channel and into
the main channel; l) supplying a gas to the gas inlet on the second
branch channel and opening the gas inlet valve on the second branch
channel; and m) passing a gas from the gas inlet on the second
branch channel through the second branch channel and into the main
channel to evacuate any residual liquid in the second branch
channel.
22. The method of claim 21, wherein steps (l) and (m) further
comprise supplying a gas to the gas inlet on the first branch
channel and opening the gas inlet valve on the first branch channel
and passing a gas from the gas inlet on the first branch channel
through the first branch channel and into the main channel to
evacuate any residual liquid in the first branch channel.
23. The method of any preceding claim, wherein the fluidic
cartridge further comprising a pneumatic interface for connecting
to a source of positive pressure, and wherein the or each gas inlet
on the one or more branch channels is coupled to the pneumatic
interface for connection to a gas supply, wherein one or more of
the steps of passing a gas from the gas inlet comprises passing a
gas from the supply of positive gas pressure into the branch
channel.
24. The method of any one of claims 19 to 23, wherein one or more
of the steps of passing a gas from the gas inlet on the one or more
branch channels and into the main channel further comprises purging
the residual liquid sample and/or residual liquid from the main
channel.
25. The method of any one of claims 19 to 23, wherein the or each
liquid inlet on the one or more branch channels is coupled to a
liquid chamber, and wherein one or more of the steps of passing a
liquid from the liquid inlet comprises expelling the liquid from
the liquid chamber into the branch channel.
26. The method of claim 25, wherein the or each liquid chamber is a
collapsible blister, and wherein one or more of the steps of
passing a liquid from the liquid inlet comprises collapsing the
collapsible blister and thereby ejecting liquid contents through
the liquid inlet, into the branch channel and into the main
channel.
27. The method of claim 25 or claim 26, when dependent on claim 21
or any claim dependent on claim 21, wherein the step of passing a
liquid from the liquid inlet on the first branch channel comprises
expelling a wash buffer from the liquid chamber through the liquid
inlet into the first branch channel and into the main channel; and
wherein the step of passing a liquid from the liquid inlet on the
second branch channel comprises expelling an elution buffer from
the liquid chamber through the liquid inlet into the second branch
channel and into the main channel.
28. The fluidic cartridge of any preceding claim, comprising
polypropylene.
29. The fluidic cartridge of any preceding claim, wherein the main
channel and the one or more branch channels are formed in a fluidic
layer of the fluidic cartridge, said fluidic layer comprising
polypropylene.
30. The fluidic cartridge of claim 17 or any claim dependent on
claim 17, wherein the fluid passageway and valve chamber are formed
in a pneumatic layer of the fluidic cartridge, said pneumatic layer
comprising polypropylene.
31. The fluidic cartridge of claim 16 or any claim dependent on
claim 16, wherein the flexible membrane comprises a thermoplastic
elastomer.
Description
FIELD
[0001] The present invention relates to a fluidic cartridge and a
method for processing a liquid sample, more particularly where the
fluid cartridge comprises a main channel and one or more branch
channels that join the main channel.
BACKGROUND
[0002] Sample preparation and analysis presents many logistical
problems. Conventionally, many medical samples (such as blood,
saliva, urine and swab eluate) are provided to a doctor, for
example a general practitioner doctor (GP) or a principle care
physician (PCP), in a local surgery without the equipment necessary
to analyse the sample. Hence, the sample must be sent to a
laboratory where the sample is analysed. The test results must then
be collated and returned to the GP to analyse the results and make
a diagnosis. This approach is inadequate. Firstly, there is a
significant risk that a sample is lost in transit or mismatched
with the wrong patient. Moreover, whilst recent developments in
technology have reduced the overall time taken to conduct the test,
the delay involved in sending the sample to a laboratory is
unsatisfactory.
[0003] Nevertheless, analytical systems of the kind found in
laboratories are complex and it is often difficult to provide
sufficient amounts of pure targets from source samples to reliably
perform downstream analytical assays. This typically prohibits
local GP surgeries from being able to carry out such tests on
site.
[0004] However, in recent years efforts have been made to reduce
the scale of the analytical systems to make tests faster and
simpler to run, and require smaller quantities of sample. For
instance, "laboratory on a chip" (LOC) devices (a subset of
microfluidic devices) integrate almost all medical tests or
diagnostic operations performed in a hospital on a single
microfluidic chip. The channels forming such microfluidics devices
handle small fluid volumes and are connected together so as to
achieve a desired function such as mixing of a sample, moving the
sample through the device, reacting the sample with different
reagents, and so on. These chips may be inserted into machines to
control the performance of a test and measure the results.
[0005] However, it has been found that handling a sample in a
microfluidics device can be very difficult. In such small channels
as are found on a conventional LOC, it is difficult to apply
external forces to move the sample from one site to another to
perform different actions on the sample. There is also a limit to
the complexity of a LOC device which operates purely using
capillary action. Furthermore, owing to the small sample sizes of
LOC's, the devices have reduced sensitivity and the probability of
a target being present in the sample is thus reduced.
[0006] An alternative approach is to use a fluidic cartridge. The
scale of the components of a fluidic cartridge is larger than for a
microfluidic device, and so it becomes possible to move a sample
through various different sites to perform different actions on it.
This makes it possible to perform more complex tests than may be
conducted using typical LOC devices, whilst still providing an
analytical system of potential use in a local GP surgery.
[0007] Scientific assays useful in medical diagnostics have
increasingly involved biochemical procedures, such as the
polymerase chain reaction ("PCR"). The PCR assay has provided a
powerful method of assaying for the presence of defined segments of
nucleic acids. It is therefore desirable to perform a PCR assay on
a fluidic cartridge.
[0008] Reducing PCR to the microchip level is important for
portable detection technologies and high-throughput analytical
systems. The method can be used to assay body fluids for the
presence of nucleic acid specific for particular pathogens, such as
the Chlamydia trachomatis bacterium, HIV or any other pathogenic
microbe.
[0009] The introduction of commercially available automated DNA
amplification assays has allowed more laboratories to introduce
these technologies for routine testing of specimens. However, there
is a need to improve the fluidic devices used for this purpose.
[0010] Fluidic devices are often used for sample preparation and
analysis of biological or chemical liquid samples. During sample
preparation, the sample typically enters through a sample input
port and may pass along a main channel or into a cavity before
reaching a sample chamber where it may be analysed. Additional
reagents, buffers, solutions or fluids may be passed along the
channel to prepare the sample for analysis. For example, when
preparing a bacterial sample for PCR analysis, a lysis buffer may
be used to lyse the bacteria, then a wash buffer may be passed
through to wash any unwanted sample matrix through to a waste
receptacle, and then the sample may be re-suspended in a final
elution buffer ready for PCR amplification.
[0011] A problem arises when automating this system, since all the
reagents needed in the reaction chamber for the sample analysis,
such as PCR amplification, should be contained on one platform in
which the sample can be inserted in a controlled operation. WO
97/16561 provides an assay system which comprises a first assembly
comprising a reaction chamber, a second assembly comprising a heat
source and a third assembly comprising a plurality of fluid
chambers. The assemblies can move with respect to each other by
sliding or translocating, so that, for example, fluid communication
between the assemblies results when the first and third assemblies
are adjoined. The carousel as described in Example 2 of WO 97/16561
includes seventeen fluid chambers and when rotated, these fluid
chambers align with the reaction chamber so as to avoid cross
contamination of the reagents. It requires that the chambers align
perfectly so as to allow fluid communication and that there is
sufficient volume of wash buffers and cleaning solutions to ensure
trace amounts of impurities are removed.
[0012] In many biological analytical systems, in particular, in
systems using amplification assays, it is important to remove
certain reagents from the system prior to conducting the analysis.
For example, it can be extremely important to remove all traces of
the lysis buffer prior to conducting PCR amplification.
Furthermore, where the microfluidic device may be re-used for a
different sample it is important to ensure the device is cleaned to
avoid cross-contamination. WO 2010/149995 discloses the use of
cleaning solutions (such as a detergent, complexing agent, alkali
or acid) or gas flushes to flush the reaction cavity prior to
reuse. The air or gas may further be used to dry the channel or
cavity between fluid passes. A single, overly long fluid path
length requires high pressures to push the sample and reagents
through the channel and also needs large volumes of cleaning
solutions to ensure the whole length of the channel is cleaned
effectively to avoid cross-contamination risks between fluid
passes.
[0013] An alternative design, to avoid using a single fluid channel
is described in WO 03/078065 and WO 2009/108260 and uses a main
channel with several intersecting channels. These channels may be
connected to separate input ports, offset from the main channel,
and provide an alternative design for fully automated systems
wherein all the reagents are contained on the microfluidic device
at the start of use. The channels may alternatively connect to
waste reservoirs and pass through analysis regions such as in WO
03/078065. WO 2009/108260 describes flushing cleaning solutions
through the main channel to clean the microfluidic circuit and
using air to push the sample through the main channel.
[0014] The introduction of secondary channels intersecting a main
channel, whilst beneficial in introducing different fluids or
reagents through separate, distinct channels, will always create
`dead-legs`. A dead-leg is a section of the channel through which
fluid does not flow, and is considered to be a source of
contamination. They are often found where a side channel intersects
a main channel since fluid can accumulate at or near the
intersection point, at the dead-leg, and remain there until the
next fluid pass, adding a contamination risk.
[0015] For instance, FIG. 16 shows a known microfluidic junction
B110, an outlet channel B111, and a plurality of circuit units
B112, B113, B114. A microfluidic junction B110 is an area for
converging multiple fluids. An outlet channel B111 is capable of
receiving fluid from the microfluidic junction B110. An outlet
channel B111 includes a first end connected with the microfluidic
junction B110, a second end connected with a waste reservoir B115,
and an analysis region B116 positioned between the first end and
the second end of the outlet channel B111. Each circuit unit
includes a source channel B117 with a first end capable of
receiving sample fluid and a second end connected with the
microfluidic junction B110; a branch channel B110 connected with
the source channel B117 at an intersection B119; and a flow
diversion system capable of differentially directing fluid flowing
through a source channel either into the microfluidic junction B110
or into a branch channel B118. The branch channel B118 is further
connected to a waste reservoir B120. When fluid enters into the
circuit units B112, the fluid flows along the source channel B117,
to the intersection B119 and is directed to either continue through
the source channel B117 and into the microfluidic junction B110, or
into the branch channel B118. Fluids that are directed toward the
junction B110 converge in the junction B110 and flow into the
outlet channel B111, through the analysis region B116 and into the
waste reservoir B115.
[0016] In FIG. 16, several dead-legs B108 exist at the intersection
of the branch channels B118 with the source channels B117, and at
the intersection of the source channel B117 with the outlet channel
B111. FIG. 17 further illustrates a series of branch channels B101,
B102, joining a main channel B100 and the presence of dead-legs
B108 in the branch channels B101 and B102 at or near these
intersection points.
[0017] It is very difficult to remove the presence of dead-legs
completely. Special valves can be designed to reduce the dead-leg,
but there is still a small but finite volume associated with these
valves which may allow for fluid accumulation. Accordingly, there
is a need in the art to provide a microfluidic device which allows
for the addition of reagents from secondary channels to the main
channel without cross-contamination.
SUMMARY OF INVENTION
[0018] According to a first aspect of the invention, there is
provided a fluidic cartridge for processing a liquid sample,
comprising: a main channel for passing the liquid sample
therethrough from an upstream end to a downstream end; and one or
more branch channels that join the main channel for introducing
liquid and gas into the main channel after the liquid sample has
passed downstream of the one or more branch channels, the one or
more branch channels including a first branch channel, wherein the
first branch channel comprises: a gas inlet for introducing a gas
into the first branch channel; a liquid inlet for introducing a
liquid into the first branch channel; and a valve configured to
move between a closed position in which it prevents liquid and gas
in the first branch channel from passing into the main channel and
an open position in which it permits liquid and gas in the first
branch channel to pass into the main channel.
[0019] This allows for containment of a liquid, such as a test
reagent, on the fluidic cartridge at the start of use and allows
for the introduction of the liquid from a channel independent of
the main channel, thus avoiding contamination of the main channel.
Moreover, gas may be introduced into the branch and main channels
to clear the channels and also push the liquid and sample through
the main channel.
[0020] Preferably, the one or more branch channels further
comprises a second branch channel which joins the main channel
downstream of the first branch channel, wherein the second branch
channel comprises: a gas inlet for introducing a gas into the
second branch channel; a liquid inlet for introducing a liquid into
the second branch channel; and a valve configured to move between a
closed position in which it prevents liquid and gas in the second
branch channel from passing into the main channel and an open
position in which it permits liquid and gas in the second branch
channel to pass into the main channel.
[0021] The second branch channel can be used to introduce a
different liquid or reagent into the main channel, and is disposed
so that the second liquid can be introduced after the passage of a
first liquid from the first branch channel.
[0022] The or each gas inlet on the one or more branch channels
preferably comprises a gas inlet valve to prevent liquid and gas
flowing from the branch channel through the gas inlet. The gas
inlet valve reduces the risk of contamination of the gas inlet by
the liquid from the liquid inlet.
[0023] Preferably, the or each valve in the one or more branch
channels is spaced from the main channel, thereby forming a
dead-leg in the branch channel between the valve and the main
channel. The introduction of secondary channels intersecting a main
channel will typically create `dead-legs`. These dead-legs act as a
contamination risk. The presence of the gas inlet and gas inlet
valve on the one or more branch channels provides a method to
overcome that risk by ensuring that the channels can be cleared of
liquid sample and/or liquid before each pass of the next
liquid.
[0024] The or each valve in the one or more branch channels may be
located at a junction between the branch channel and the main
channel. This can reduce the length of the branch channel (as it is
not required to accommodate an additional valve), and thus save
space on the fluidic device.
[0025] Preferably, the or each liquid inlet on the one or more
branch channels is coupled to a liquid chamber. The chamber
contains the liquid on the fluidic cartridge, and prevents leakage
of the liquid which would detrimentally affect the use of the
device.
[0026] Preferably, the or each liquid chamber is a collapsible
blister adapted, when it is collapsed, to eject a liquid contained
therein through the liquid inlet, into the branch channel, and into
the main channel, for introducing the liquid into the main channel
after the liquid sample has passed downstream of the one or more
branch channels. The blister contains the liquid and provides a
mechanism for releasing the liquid into the fluidic device during
operation that does not involve externally introducing liquid
components.
[0027] The or each liquid chamber preferably contains a reagent or
a buffer such as a lysis buffer, a wash buffer or an elution
buffer. These reagents may be capable of performing cell lysis and
cleaning the sample as well as ensuring the sample is prepared for
analysis.
[0028] Preferably, wherein a first and a second branch channel join
the main channel, the liquid chamber coupled to the liquid inlet in
the first branch channel contains a wash buffer, and wherein the
liquid chamber coupled to the liquid inlet in the second branch
channel contains an elution buffer. Since wash buffers are `toxic`
to subsequent downstream reactions (in particular, they prevent the
action of elution buffers) the wash buffer is passed first and then
the branch and main channels can be cleaned/evacuated with a gas
pass. The elution buffer can then pass through the channels and
downstream to the liquid sample.
[0029] Preferably, the fluidic cartridge comprises a pneumatic
interface for connecting to a source of positive and/or gauge
pressure, the pneumatic interface comprising a plurality of ports,
and wherein the or each valve or gas inlet valve in the one or more
branch channels is a pneumatically-actuated valve coupled to at
least one port in the pneumatic interface such that it may be
actuated by the source of positive and/or gauge pressure.
Preferably, the first and second gas inlet valves in the first and
second branch channels, respectively, are coupled to the same port
in the pneumatic interface, such that the first and second gas
inlet valves may be actuated simultaneously. This simplifies the
operation of the cartridge.
[0030] The or each gas inlet on the one or more branch channels is
preferably coupled to the pneumatic interface for connection to a
gas supply.
[0031] Preferably, the or each gas inlet on the one or more branch
channels is coupled to the pneumatic interface for connection to a
gas supply for passing a gas through the gas inlet, into the branch
channel, and into the main channel, for introducing the gas into
the main channel after the liquid sample has passed downstream of
the one or more branch channels. The passing of gas through the
branch and main channels effectively evacuates the dead-legs and
cleans the channels. The gas can further dry the channels of liquid
residues.
[0032] Preferably, the or each pneumatically-actuated valve
comprises a valve chamber having first and second openings
connected to the branch channel; and a flexible membrane movable
between a closed position, in which the flexible membrane seals
against the first and second openings to prevent fluid flow through
the branch channel, and an open position, in which the flexible
membrane is spaced apart from the first and second openings to
permit fluid to flow through the branch channel.
[0033] Preferably, the or each valve further comprises a fluid
passageway having an opening in the valve chamber, the opening
separated from the first and second openings by the flexible
membrane, wherein the fluid passageway is coupled to a port in the
pneumatic interface for applying a positive or gauge pressure in
the valve chamber to move the flexible membrane between the open
and closed positions. The fluid passageway in the valve chamber
controls the pressure of the chamber, enabling the flexible
membrane to act as a valve.
[0034] Preferably, the pneumatic interface comprises first to third
ports, and wherein the first to third ports are respectively
coupled to: i) the valve in the first branch channel; ii) the valve
in the second branch channel; and iii) the gas inlet valve in the
first branch channel and the gas inlet valve in the second branch
channel; such that the respective valves may be actuated by the
source of positive and/or gauge pressure acting through the
respective ports. The coupling of the gas inlet valves on the first
and second branch channels enables control of these valves
simultaneously.
[0035] According to a second aspect of the invention, there is
provided a method of processing a liquid sample in a fluidic
cartridge, the cartridge comprising a main channel and one or more
branch channels that join the main channel, including a first
branch channel comprising a gas inlet, a liquid inlet and a valve,
the method comprising; a) passing a liquid sample through the main
channel; b) supplying a gas to the gas inlet; c) opening the valve
on the first branch channel and passing a gas from the gas inlet
through the first branch channel and into the main channel to
evacuate any residual liquid sample in the first branch channel; d)
ceasing the supply of gas; e) passing a liquid from the liquid
inlet through the first branch channel and into the main channel;
f) supplying a gas to the gas inlet; and g) passing a gas from the
gas inlet through the first branch channel and into the main
channel to evacuate any residual liquid from the first branch
channel. This method is particularly effective since the user can
rely on the passing of gas through the branch and main channels to
evacuate any dead-legs that are present, as well as clearing any
residual liquid sample or liquid in the channels.
[0036] Preferably, the fluidic cartridge further comprises a gas
inlet valve on the first branch channel, and the method steps (b)
and (f) of supplying a gas to the gas inlet further comprise
opening the gas inlet valve, and wherein step (d) of ceasing the
supply of gas further comprises closing the gas inlet valve and
before ceasing the supply of gas. This prevents liquid from passing
from the liquid inlet and into the gas inlet when a liquid is
ejected from the liquid chamber.
[0037] According to a further aspect of the invention, there is
provided a method of processing a liquid in a fluidic cartridge,
the cartridge comprising a main channel having an upstream end and
a downstream end and one or more branch channels that join the main
channel, including a first branch channel and second branch channel
downstream of the first branch channel, each branch channel
comprising a gas inlet, a gas inlet valve, a liquid inlet and a
valve, the method comprising: a) passing a liquid sample through
the main channel; b) supplying a gas to the gas inlet on the first
branch channel and the gas inlet on the second branch channel; c)
opening the valves and the gas inlet valves on the first and second
branch channels and passing a gas from the gas inlets on the first
and second branch channels through the first and second branch
channels and into the main channel to evacuate any residual liquid
sample in the first and second branch channels; d) closing the
valve and the gas inlet valve on the second branch channel and the
gas inlet valve on the first branch channel; e) ceasing the supply
of gas to the gas inlet on the first branch channel and the gas
inlet on the second branch channel; f) passing a liquid from the
liquid inlet on the first branch channel through the first branch
channel and into the main channel; g) supplying a gas to the gas
inlet on the first branch channel and the gas inlet on the second
branch channel; h) opening the valve and the gas inlet valve on the
second branch channel and the gas inlet valve on the first branch
channel and passing a gas from the gas inlets on the first and
second branch channels through the first and second branch channels
and into the main channel to evacuate any residual liquid in the
first and second branch channels; i) closing the valve and the gas
inlet valve on the first branch channel and the gas inlet valve on
the second branch channel; j) ceasing the supply of gas to the gas
inlet on the first branch channel and the gas inlet on the second
branch channel; k) passing a liquid from the liquid inlet on the
second branch channel through the second branch channel and into
the main channel; l) supplying a gas to the gas inlet on the second
branch channel and opening the gas inlet valve on the second branch
channel; and m) passing a gas from the gas inlet on the second
branch channel through the second branch channel and into the main
channel to evacuate any residual liquid in the second branch
channel. This method allows for the passing of a liquid sample and
two liquid reagents through the main channel. The passing of the
sample and liquids in separate steps, with gas passes in between,
ensures the channels are evacuated and any dead-legs present are
cleared prior to the next liquid pass. This reduces contamination
risk.
[0038] Preferably, the method steps (l) and (m) further comprise
supplying a gas to the gas inlet on the first branch channel and
opening the gas inlet valve on the first branch channel and passing
a gas from the gas inlet on the first branch channel through the
first branch channel and into the main channel to evacuate any
residual liquid in the first branch channel. In the unlikely event
that elution buffer may have passed upstream, this step ensures
that the first branch channel is also cleared of any elution
buffer, which is of particular importance when sample and reagent
volumes are being controlled.
[0039] When in use, the fluidic cartridge may preferably further
comprise a pneumatic interface for connecting to a source of
positive pressure, and wherein the or each gas inlet on the one or
more branch channels is coupled to the pneumatic interface for
connection to a gas supply, wherein one or more of the steps of
passing a gas from the gas inlet comprises passing a gas from the
supply of positive gas pressure into the branch channel.
[0040] Preferably, the one or more of the steps of passing a gas
from the gas inlet on the one or more branch channels and into the
main channel further comprises purging the residual liquid sample
and/or residual liquid from the main channel. This ensures the
total volume of liquid sample and/or liquid is passed to the
downstream end of the main channel, which, as stated previously, is
important when the liquid volume is to be precisely controlled.
[0041] Preferably, the or each liquid inlet on the one or more
branch channels is coupled to a liquid chamber, and wherein one or
more of the steps of passing a liquid from the liquid inlet
comprises expelling the liquid from the liquid chamber into the
branch channel. The or each liquid chamber may preferably be a
collapsible blister, and wherein one or more of the steps of
passing a liquid from the liquid inlet comprises collapsing the
collapsible blister and thereby ejecting liquid contents through
the liquid inlet, into the branch channel and into the main
channel.
[0042] Preferably, the step of passing a liquid from the liquid
inlet on the first branch channel comprises expelling a wash buffer
from the liquid chamber through the liquid inlet into the first
branch channel and into the main channel; and wherein the step of
passing a liquid from the liquid inlet on the second branch channel
comprises expelling an elution buffer from the liquid chamber
through the liquid inlet into the second branch channel and into
the main channel. These steps ensure the wash buffer and elution
buffer are passed independently to the downstream end of the main
channel, and thus avoids any contamination of the buffers (which
would result in the elution buffer becoming inactive).
BRIEF DESCRIPTION OF THE FIGURES
[0043] FIG. 1 is a schematic diagram of an exemplary fluidic
cartridge in which the invention may be provided.
[0044] FIG. 2 is a top view of an exemplary fluidic cartridge in
which the invention may be provided.
[0045] FIG. 3 is an exploded view of the exemplary fluidic
cartridge of FIG. 2.
[0046] FIG. 4 is a perspective view of the housing of the exemplary
fluidic cartridge of FIG. 2.
[0047] FIG. 5 is a perspective view of the blister sub-assembly of
the exemplary fluidic cartridge of FIG. 2.
[0048] FIG. 6A is a top view of the pneumatic layer of the
exemplary fluidic cartridge of FIG. 2.
[0049] FIG. 6B is a bottom view of the pneumatic layer of the
exemplary fluidic cartridge of FIG. 2.
[0050] FIG. 7 is a top view of the pneumatic foil of the exemplary
fluidic cartridge of FIG. 2.
[0051] FIG. 8A is a top view of the fluidic layer of the exemplary
fluidic cartridge of FIG. 2.
[0052] FIG. 8B is a bottom view of the fluidic layer of the
exemplary fluidic cartridge of FIG. 2.
[0053] FIG. 9 is a top view of the fluidic foil of the exemplary
fluidic cartridge of FIG. 2.
[0054] FIG. 10 is a top view of the electrode layer of the
exemplary fluidic cartridge of FIG. 2.
[0055] FIG. 11 is a section view of an advantageous valve
arrangement which may form an isolated inventive aspect.
[0056] FIG. 12 is a section view of another advantageous valve
arrangement which may form an isolated inventive aspect.
[0057] FIG. 13a is a section view of an advantageous inlet port
arrangement which may form an isolated inventive aspect.
[0058] FIG. 13b is a perspective section view of the inlet port
arrangement of FIG. 13a.
[0059] FIG. 14a is a section view of an advantageous capture column
arrangement which may form an isolated inventive aspect.
[0060] FIG. 14b is a perspective section view of a portion of the
capture column arrangement of FIG. 14a.
[0061] FIG. 15a is a section view of an advantageous waste chamber
arrangement which may form an isolated inventive aspect.
[0062] FIG. 15b is a perspective section view of the waste chamber
arrangement of FIG. 15a.
[0063] FIG. 16 in an exemplary system of interconnecting channels
showing the problem of dead-legs.
[0064] FIG. 17 is a further illustration of channels showing the
problem of dead-legs.
[0065] FIG. 18a is a schematic diagram of channels within a fluidic
cartridge according to a first embodiment of the present
invention.
[0066] FIG. 18b is an alternative arrangement of the first
embodiment shown in FIG. 13a.
[0067] FIG. 19a is a schematic diagram of channels within a fluidic
cartridge according to a second embodiment of the present
invention.
[0068] FIG. 19b is an alternative arrangement of the second
embodiment shown in FIG. 14a.
[0069] FIG. 19c is another alternative arrangement of the second
embodiment shown in FIG. 14a.
[0070] FIG. 20a is a section view of a blister suitable for use in
the present invention.
[0071] FIG. 20b is a top view of the blister shown in FIG. 15a.
[0072] FIG. 21a is a section view of a valve suitable for use in
the present invention, wherein the valve is in the closed
position.
[0073] FIG. 21b is a section view of the valve of FIG. 16a wherein
the valve is in the open position.
[0074] FIG. 22 is a schematic diagram of channels within a fluidic
cartridge according to a third embodiment of the present
invention.
[0075] FIG. 23 is a schematic diagram of channels within a fluidic
cartridge according to a fourth embodiment of the present
invention.
[0076] FIG. 24 is flow diagram showing steps in a method of
operating the cartridge shown in FIG. 18a.
[0077] FIG. 25 is flow diagram showing steps in a method of
operating the cartridge shown in FIG. 19a.
DETAILED DESCRIPTION
[0078] Embodiments of the invention will now be described in the
context of an exemplary fluid cartridge in which the invention is
implemented. Whilst not necessary to understand the present
invention, it is beneficial to provide general description of the
principles of the structure, manufacture, function and use of the
fluidic cartridge and associated methods for performing a test.
[0079] The exemplary fluidic cartridge and associated methods
chosen to illustrate the present invention are for the detection of
Chlamydia trachomatis bacterium using PCR amplification and
electrochemical detection. However, the skilled person would
understand that the invention is not limited to the exemplary
fluidic cartridge and associated methods, and is suitable for use
in with various different cartridges for a wide variety of sample
analysis techniques or biological assays; for example, assays of
target nucleic acid sequences in a liquid sample.
[0080] Those skilled in the art will understand that the devices
and methods of the invention described herein and illustrated in
the accompanying drawings are non-limiting exemplary embodiments
and that the scope of the present invention is defined solely by
the claims. The features illustrated or described in connection
with one exemplary embodiment may be combined with features of
other embodiments. Such modifications and variations are included
within the scope of the present disclosures.
[0081] The exemplary cartridge comprises: a fluidic portion through
which the sample flows and in which nucleic acid amplification and
detection take place; a pneumatic portion which controls flow
through the fluidic portion; and at least two electrodes which
provide a potential difference for the detection of an amplified
nucleic acid of interest. The fluidic portion and pneumatic portion
may be constructed of a fluidic layer, a fluidic foil, a pneumatic
layer and a pneumatic foil such as those described in relation to
the exemplary cartridge below. However, the fluidic portion does
not necessarily consist only of a fluidic layer and a fluidic foil
and the pneumatic portion does not necessarily consist only of a
pneumatic layer and a pneumatic foil. Rather, the layers may
interact to produce the fluidic portion and the pneumatic portion
such that parts of all or some of the layers make up each portion.
Rather than referring to the particular layers of the cartridge,
the fluidic portion refers to the particular areas of the cartridge
which provide the function of allowing controlled sample flow, and
the pneumatic portion refers to the particular areas of the
cartridge which provide the function of controlling the flow
through the fluidic portion.
[0082] The housing, fluidic portion and pneumatic portion are made
of plastic. By plastic is meant a synthetic or natural organic
material that may be shaped when soft and then hardened, including
resins, resinoids, polymers, cellulose derivatives, casein
materials, and protein plastics. Examples of plastics from which
the cartridge may be constructed include, but are not limited to
thermoplastics, for example polycarbonate, polyethylene
terephthalate, cyclic olefin copolymers such as Topaz,
acrylonitrile butadiene styrene, and thermoplastic elastomers, for
example polypropylene. Plastic housings, fluidic portions and
pneumatic portions can include components which are not made of
plastic (e.g. blisters made from metal foil, metallic inserts at
the sample inlet), but they are formed primarily from plastic. The
use of plastic materials facilitates economical manufacture of the
cartridges.
[0083] Whilst the pneumatic and fluidic foils may be made from a
metal foil, the preferred materials are plastic including those
mentioned above. In particular, it is preferred that foils are a
polyethylene terephthalate/polypropylene composite.
[0084] The target nucleic acid sequence is any nucleic acid to be
detected in a sample. The target nucleic acid(s) to be amplified
and detected in the cartridge will usually be DNA, but it is also
possible to amplify and detect RNA. In some embodiments a cartridge
may permit amplification and/or detection of both DNA and RNA
targets.
[0085] The liquid sample is the composition which is introduced
into the cartridge in order to determine whether the target nucleic
acid(s) of interest is/are present. The sample may be a composition
in which the nucleic acid to be detected is suspected to be present
(e.g. for clinical diagnosis), or may be a composition in which the
nucleic acid to be detected is potentially present (e.g. for
contamination testing).
[0086] The liquid sample can have various sources. For instance, it
can be material obtained from an animal or plant (e.g. for
diagnosis of infections or for genotyping). Such samples may be
obtained with minimal invasiveness or non-invasively, e.g., the
sample may be obtained from an animal using a swab, or may be a
bodily fluid. As an alternative, the sample may be material
obtained from food or water (e.g. for contamination testing). The
sample will usually include cells, and the target nucleic acid (if
present) can be extracted from these cells within the cartridge.
One skilled in the art will appreciate that samples can be diluted
or otherwise treated prior to being introduced into the cartridge,
but it is preferred that the cartridge can handle material which
has not been pre-treated in this way.
[0087] An animal from whom the sample is obtained may be a
vertebrate or non-vertebrate animal. Vertebrate animals may be
mammals. Examples of mammals include but are not limited to mouse,
rat, pig, dog, cat, rabbit, primates or the like. The animal may be
a primate, and is preferably a human. Thus the cartridge can be
used for clinical diagnosis of human samples.
[0088] In addition to analysing a sample, the cartridge can analyse
a positive and/or negative control to provide confirmation that the
cartridge is functioning as expected. The control(s) can be
introduced into the cartridge by a user, or can be included within
a cartridge before use.
[0089] The inclusion of an internal positive control nucleic acid
allows a user to identify whether a negative result for the sample
has been obtained because the nucleic acid amplification has been
unsuccessful (false negative). If the positive control nucleic acid
fails to be detected in the detection chamber, despite its presence
in an amplification chamber, the user will be able to identify the
test as a potential false negative result, and can perform another
test.
[0090] The inclusion of an internal negative control allows a user
to identify whether a positive result has been falsely obtained
because of the presence of contamination. A negative control can
involve performing PCR in a chamber in which no nucleic acid is
provided, or in which a sample undergoes an amplification reaction
without necessary components e.g. PCR without primers. If nucleic
acid is nevertheless detected in the detection chamber, despite its
intended absence in an amplification chamber, the user will be able
to identify the test as a potential false positive result, and can
perform another test.
[0091] A positive control nucleic acid may be any nucleic acid that
will not be found in a sample used in the cartridge. The internal
control DNA may be taken from a bacterium that is not pathogenic to
animals and which contains a nucleic acid that is highly specific
to the bacterium. One example of a possible bacterium from which
the control nucleic acid may be taken for an animal sample is
Pectobacterium atrosepticum, although any control nucleic acid may
be used that will not be present in a sample.
[0092] The fluidic portion of the cartridge comprises channels and
chambers through which sample flows. The flow of sample through the
cartridge is controlled in two ways. Firstly, the fluidic portion
has a gas inlet. The gas inlet is connected to a gas supply, and
injection of gas into the fluidic portion via this inlet allows the
sample to be pushed downstream through the cartridge, towards the
detection chamber. The gas supply may be provided by the reader. As
an alternative, the gas supply may be an on-board gas supply.
Preferably, the gas supply is provided by an external source and
the gas inlet is connected to a pneumatic circuit such that the gas
supply is provided via a pneumatic inlet on the cartridge.
Secondly, at least one pneumatically controlled valve controls
local movement of the sample through the fluidic portion. The
pneumatically controlled valve(s) may be controlled independently
of other pneumatically controlled valves and may be controlled
independently of the gas supply that generally causes downstream
movement of the sample via the gas inlet. The gas inlet and the
pneumatically controlled valve(s) also permit sample to be flushed
through the fluidic portion e.g. to exclude excess volumes of
material. The fluidic portion also has an exhaust which allows air
and waste material to exit the channels and chambers of the fluidic
portion without a build-up of pressure occurring in the cartridge.
Preferably, the exhaust comprises a waste chamber and/or a waste
vent.
[0093] The fluidic portion of the cartridge includes reagents
and/or physical components for cell lysis and nucleic acid
separation. These may be any reagents or physical components that
are capable of lysing cells and separating nucleic acids from cell
debris and other cellular components. For instance, they may
comprise (i) a lysis buffer which is capable of causing lysis of
target cells which may be present in the sample e.g. buffers
including a detergent such as nonyl phenoxypolyethoxylethanol
(available as NP-40) or t-octylphenoxypolyethoxyethanol, (available
as Triton X 100), or including guanidine thiocyanate, and/or (ii) a
capture support or column which specifically binds nucleic acids
but does not bind other undesired cellular components (e.g.
proteins and lipids). The capture column comprises a capture filter
and may additionally comprise a depth filter. The filters may be
made of glass fibres (available as Whatman filters), or may be made
of silica, although any column or support which is capable of
separating nucleic acids from other cellular components may be
used. Elution using a wash buffer to remove cell debris and other
cellular components, followed by elution using an elution buffer to
elute the separated nucleic acids from the capture support or
column can be undertaken such that the capture column can separate
nucleic acids from cell debris and other cellular components.
[0094] A channel through which the sample flows fluidly connects
the sample inlet to at least one amplification chamber where
nucleic acid amplification can take place. The purpose of the
amplification chamber(s) is to permit amplification of any target
nucleic acid of interest that is present in the sample (and, where
present, any positive control nucleic acid). Any nucleic acid
amplification method may be used and these are described in more
detail below in relation to an exemplary cartridge. The different
nucleic acid amplification reagents that are required for different
nucleic acid amplification methods are well known in the art. These
reagents are provided in or upstream of the amplification
chamber(s) such that the sample (and any positive control) includes
all necessary reagents for nucleic acid amplification once it
reaches the amplification chamber. Adaptation of a nucleic acid
amplification method according to the target nucleic acid to be
detected is also well known in the art (e.g. design of primers).
The skilled person would therefore be able to adapt the reagents
for nucleic acid amplification accordingly. The term "chamber" does
not denote any particular size or geometry, but instead it means a
region within the fluidic portion which is designed to permit
nucleic acid amplification to occur. Thus, for instance, it could
be a region in which the sample can be fluidically isolated (e.g.
via the use of pneumatically controlled valves) while the steps
required for nucleic acid amplification (e.g. thermocycling, etc.)
occur, and it can be located within the cartridge so that it is in
the proximity of any external resources that are needed (e.g. next
to a heat source within a cartridge reader, thereby permitting
thermal cycling to occur).
[0095] Multiple test amplification channels and/or chambers may be
included in the cartridge. The different test amplification
channels and/or chambers may include reagents required to amplify
different nucleic acids of interest. Therefore using multiple
amplification test channels and/or chambers allows multiple tests
to be performed on a single cartridge, simultaneously (including
any controls). As an alternative, reagents for amplification of
multiple different nucleic acids may be present in a single
amplification chamber, and the different nucleic acids (whether
multiple target nucleic acids, or a target nucleic acid and a
control nucleic acid) may be amplified simultaneously in the same
amplification chamber.
[0096] A further channel through which the sample flows after
nucleic acid amplification fluidly connects the at least one
amplification chamber to at least one detection chamber where the
results of nucleic acid amplification can be detected. In or
upstream of the detection chamber are reagents for nucleic acid
detection such that the sample includes all necessary reagents for
the detection once it reaches the detection chamber. The reagents
for nucleic acid detection may be specific for the particular
target nucleic acid, i.e. they may allow for detection of the
presence of the specific nucleic acid sequence. As an alternative,
the reagents for nucleic acid detection may be generic reagents to
detect the presence of any nucleic acids. Such generic reagents may
be used if all nucleic acids other than the target nucleic acid are
removed prior to detection. For example, this may be achieved by
providing a nuclease that is capable of hydrolysing all nucleic
acids present in the sample other than the target nucleic. The
amplified target nucleic acid can be protected from hydrolysis, for
example by inclusion of chemical modifications in the primers which
are incorporated into the amplified product and which cannot be
hydrolysed. Reagents for nucleic acid detection are described below
in relation to an exemplary cartridge but usually comprise a probe
including a label. The probe is capable of hybridising to the
amplified nucleic acid which has been amplified in the
amplification chamber(s). Following hybridisation of the probe to
the amplified nucleic acid, the detection of the nucleic acid may
occur via a detectable change in the signal from the label. In some
embodiments the change may be caused by hydrolysis of the probe.
Where the probe is hydrolysed, hydrolysis is usually achieved using
a double strand specific nuclease, which can be an exonuclease or
an endonuclease. Preferably, the nuclease is T7 endonuclease. The
signal from the label is capable of undergoing a change following
hydrolysis of the probe. This is due to a change in the environment
of the label when it moves from being bound to the rest of the
probe to being free from the rest of the probe or bound to a single
nucleotide or a short part of the probe. Further details of the
types of probes and detection methods that may be used can be found
in Hillier et al. Bioelectrochemistry, 63 (2004), 307-310. As an
alternative, methods for causing a detectable change in the signal
from the label which do not rely on hydrolysis of the probe may be
used e.g. see Ihara et al. Nucleic Acids Research, 1996, Vol. 24,
No. 21 4273-4280. This change in environment of the label leads to
a change in the signal from the label. The change in signal from
the label can be detected in order to detect the presence of the
nucleic acid of interest.
[0097] Where a positive control nucleic acid is used, the reagents
for nucleic acid detection will additionally include a positive
control probe including a label. The positive control probe is
capable of hybridising to the amplified control nucleic acid. The
signal provided by the labels of the positive control and target
probes may be the same, but present in separate detection chambers
such that the signals corresponding to the control and test nucleic
acids can be distinguished. As an alternative, the signal provided
by the labels of the control and target probes may be different,
such that the signals are distinguishable from one another, even if
the probes are present in the same detection chamber.
[0098] Multiple test detection channels and/or chambers may be
included in the cartridge. The different test detection channels
and/or chambers may include reagents required to detect different
nucleic acids of interest. Therefore using multiple detection test
channels and/or chambers allows multiple tests to be performed on a
single cartridge, simultaneously. As an alternative, reagents for
detection of multiple different nucleic acids may be present in a
single detection chamber, and the different nucleic acids (whether
multiple target nucleic acids or a target nucleic acid and a
control nucleic acid) may be detected simultaneously in the same
detection chamber.
[0099] The label is detectable by use of the cartridge's
electrodes, and so the label will usually be an electrochemical
label, such as a ferrocene. Examples of labels which may be used
can be found in WO03/074731, WO2012/085591 and PCT/GB2013/051643.
Signal emitted by the label can be detected by a cartridge
reader.
[0100] The pneumatic portion of the cartridge comprises at least
one pneumatic circuit which each control at least one pneumatically
controlled valve. The pneumatic portion controls sample flow
through the cartridge by the opening and closing of pneumatically
controlled valves. The opening and closing of the valves is
controlled by changes in pneumatic pressure in the pneumatic
circuit that is applied through a pneumatic pressure inlet.
Usually, the cartridge contains many pneumatically controlled
valves. The pneumatically controlled valves may be controlled by
separate pneumatic pressure inlets. These valves can be used to
prevent downstream movement of sample through the fluidic portion
until necessary steps have been performed and/or to prevent
unwanted reverse movement of sample upstream. For example, a valve
may be provided upstream of the at least one amplification chamber
in order to prevent downstream movement into the at least one
amplification chamber until cell lysis and nucleic acid separation
has taken place. Following cell lysis and nucleic acid separation
the valve upstream of the at least one amplification chamber may be
opened in order to allow downstream flow. It can then be closed
again, to prevent backflow out of the chamber back towards the
sample inlet.
[0101] The cartridge comprises at least two electrodes which can
provide a potential difference across the at least one detection
chamber. The potential difference causes current to flow through
the at least one detection chamber, thereby permitting the
detection of signal from electrochemically active labels.
[0102] An exemplary cartridge which operates according to the above
description will now be described with reference to the
accompanying drawings.
[0103] 1. The Exemplary Cartridge
[0104] 1.1 Overview
[0105] The exemplary cartridge described below is intended to be a
single-use, disposable cartridge for performing a test on a sample
introduced into the cartridge. The exemplary cartridge is a fluidic
cartridge with channels of an appropriate scale (as detailed
hereafter). However, the invention may be performed on a
microfluidic device, or an LOC. Once the test has been run, it is
preferred that the cartridge is disposed of. However, if desired,
the cartridge may be sent for re-processing to enable it to be used
again.
[0106] It is preferred that the cartridge comprises all of the
biological agents necessary for conducting the test of choice. For
example, the exemplary cartridge is used for detecting the
presence, absence or amount of a pathogen of interest. Any pathogen
may be detected. Examples of pathogens which may be detected by the
cartridge are Chlamydia trachomatis, Trichomonas vaginalis,
Neisseria gonorrhoea, Mycoplasma genitalium and methicillin
resistant Staphylococcus aureus. To that end the cartridge
comprises reagents for nucleic acid amplification. Nucleic acid
amplification may be performed using any nucleic acid amplification
method. The nucleic acid amplification method may be a
thermocycling method in which the temperature at which the method
is performed is varied such that different steps of the
amplification are able to take place at different temperatures
within the cycle. For example melting, annealing of primers and
extension may each be performed at different temperatures. By
cycling through the temperatures, the timing of each of the steps
of the method can be controlled. As an alternative, the nucleic
acid amplification may be an isothermal method in which the
temperature is kept constant. In both the thermocycling and the
isothermal nucleic acid amplification methods, the temperature is
controlled during nucleic acid amplification.
[0107] Examples of nucleic acid amplification methods are the
polymerase chain reaction (PCR), the ligase chain reaction (LCR),
strand displacement amplification (SDA), transcription mediated
amplification, nucleic acid sequence-based amplification (NASBA),
helicase-dependent amplification and loop-mediated isothermal
amplification. The reagents for nucleic acid amplification will
vary depending of the nucleic acid amplification method used but
include a polymerase and nucleotide triphosphates.
[0108] As explained below, the cartridge also comprises detection
reagents which are capable of detecting the presence or absence of
amplified nucleic acids which are the product of the nucleic acid
amplification method. The reagents for nucleic acid detection
comprise a probe which is capable of hybridising to the amplified
nucleic acid. The probe includes a ferrocene label.
[0109] Following hybridisation of the probe to the amplified
nucleic acid, the detection of the nucleic acid occurs via a
detectable change in the signal from the label. The change is
caused by hydrolysis of the probe, which is achieved using a double
strand specific nuclease. The nuclease is a T7 endonuclease. The
ferrocene gives different electrochemical signals when it is part
of a probe or when it is attached only to a single nucleotide, and
so hydrolysis is easily detected. Thus, the change in signal from
the label permits detection of the presence of the nucleic acid of
interest.
[0110] The electrodes allow the detectable change in the signal
from the label, which occurs in the presence of the target nucleic
acid, to be detected.
[0111] The cartridge is configured for use with a cartridge reader
(not shown). The cartridge comprises a number of pneumatic,
mechanical, thermal and electrical interfaces (described in more
detail below) through which the reader interacts with the cartridge
to perform the test. Hence, in use, the cartridge would be inserted
into the reader, and the reader would be activated to begin
interacting with the cartridge via the interfaces to perform the
test. For the purposes of understanding the present invention, it
is not necessary to describe exactly how the cartridge interacts
with the reader to conduct a particular test and provide the test
results, but an overview of an exemplary operation of a cartridge
is provided hereafter.
[0112] 1.2 Schematic Diagram of the Exemplary Cartridge
[0113] Before explaining the structure and arrangement of the
components of an exemplary fluid cartridge in detail, it is helpful
to describe the layout of the exemplary cartridge at a high level
with reference to the schematic shown in FIG. 1.
[0114] It is convenient to consider the overall layout of the
cartridge in terms of the flow of liquids, including the liquid
sample, through the cartridge. Unless otherwise specified
hereafter, the passage of liquids including the liquid sample and
the liquid buffers is referred to as the `fluid pathway` which has
an upstream end and a downstream end. Unless otherwise specified
hereafter, `downstream` generally refers to the direction of flow
of the liquids and `upstream` refers to the direction opposite the
direction of flow. The fluid pathway in the exemplary cartridge may
have different branches (and thus form different fluid pathways),
but all pathways have a recognisable direction of flow which permit
a skilled person to identify the upstream and downstream
directions. However, there is an exception to this general
definition, which is when the liquid sample is pumped between the
mixing chamber 10 and the bellows 20. In this case, fluid is
intermittently pumped back upstream in the opposite direction to
its general direction of fluid flow, which is downstream. This
mixing serves to mix the lysis and sample and to rehydrate the
internal control.
[0115] The liquid sample is introduced into the cartridge at a
sample mixing chamber 10 through an entry port. A particular
arrangement of a preferred entry port may itself form an isolated
inventive aspect of the cartridge, as described further in section
3, below. A sample indicator 12 is fluidly coupled to the sample
mixing chamber 10 such that a sample introduced into the sample
mixing chamber 10 is visible in the sample indicator 12. Also
connected to the sample mixing chamber 10 is a blister 14
containing a lysis buffer. The lysis buffer comprises guanidine
thiocyanate. Once the sample has been introduced into the sample
mixing chamber 10, and a test is started, the lysis blister 14 is
collapsed so as to expel the lysis buffer into the sample mixing
chamber 10 where it mixes with the liquid sample introduced
therein.
[0116] Downstream of the sample mixing chamber 10, along a main
channel 16, is a coarse filter 18. The coarse filter 18 filters out
any large debris in the liquid sample, such as skin or bodily hair,
as the liquid sample passes through main channel 16.
[0117] Downstream of the coarse filter 18, along the main channel
16, is a bellows 20 having an upstream bellows valve 22a and a
downstream bellows valve 22b. As described in more detail below,
the bellows 20, together with its upstream and downstream valves
22a-b, is capable of pumping the liquid sample from the upstream
end of the fluid pathway (i.e. from the sample mixing chamber 10)
to the downstream end. In summary, this is achieved by virtue of
flexible membranes within the bellows 20 and the upstream and
downstream bellows valves 22a-b which actuate to create local
pressure differentials to, on the one hand, draw in the liquid
sample from the sample mixing chamber 10 into the bellows 20 and,
on the other hand, from the bellows 20 further downstream through
the main channel 16. This is achieved by carefully choreographed
pneumatic actuation of the flexible membranes in the valves.
Particular arrangements of a preferred valve may themselves form
isolated inventive aspects of the cartridge, as described further
in section 3, below.
[0118] Downstream of the bellows along the main channel 16 is a
capture column 24. The purpose of the capture column 24 is to
separate nucleic acids from cell debris and other cellular
components. The capture column comprises a capture filter and a
depth filter both made of glass fibres. A particular arrangement of
a preferred capture column may itself form an isolated inventive
aspect of the cartridge, as described further in section 3,
below.
[0119] Two branch channels 26, 28 join the main channel 16 between
the downstream bellows valve 22b and the capture column 24. The
purpose of the branch channels is to introduce liquid buffers
necessary for performing the desired test. For example, with the
test conducted by the exemplary cartridge, it is necessary to
introduce an elution buffer and a wash buffer into the main channel
once the sample has passed through. The wash buffer is contained in
a wash buffer blister 30 and the elution buffer is contained in an
elution buffer blister 32. The introduction of the wash buffer and
elution buffer into the main channel 16 is controlled by wash
buffer valve 34 and elution buffer valve 36, respectively. At the
appropriate point in the test, the wash and elution buffer blisters
30, 32 are collapsed so as to expel the wash and elution buffers
into the branch channels 26, 28 and thence into the main channel 16
through the wash and elution buffer valves 34, 36.
[0120] Downstream of the capture column 24, along a waste branch
16a of the main channel 16, is a waste chamber 38. A particular
arrangement of a preferred waste chamber may itself form an
isolated inventive aspect of the cartridge, as described further in
section 3, below. The purpose of the waste chamber 38 is to collect
the cell debris and cellular components other than nucleic acids
and contain them, thereby preventing them from entering the test
channel 54a or the control channel 54b. The waste chamber 38 is
vented to atmosphere through a waste vent 40, and an aerosol
impactor 42 is provided between the waste chamber 38 and the waste
vent 40 to prevent particulate matter from escaping from the waste
chamber 38 into the atmosphere. A waste chamber valve 44 in the
main channel waste branch 16a of the main channel 16 permits and
prevents fluids passing into the waste chamber 38 at appropriate
points during the test.
[0121] Downstream of the capture column 24, along an elution branch
16b of the main channel 16, is an elution chamber 46. The purpose
of the elution chamber 46 is to allow the sample preparation to
settle and for bubbles to disperse before the sample enters the
amplification chambers. An elution chamber valve 48 in the elution
branch 16b of the main channel 16 permits and prevents fluids
passing into the elution chamber 46 at appropriate points during
the test.
[0122] Downstream of the elution chamber 46 is a convoluted mixing
channel 52. Here the prepared sample is mixed prior to passing
through the isolation valve 50.
[0123] In the present application, the components upstream of the
isolation valve 50 are referred to as being comprised in the `front
end` of the cartridge, whilst the components downstream of the
isolation valve 50 are referred to as being comprised in the `back
end` of the cartridge. Broadly speaking, the liquid sample is
prepared for analysing in the front end of the cartridge, and the
analysis is carried out on the sample in the back end of the
cartridge.
[0124] The isolation valve 50 is open to permit the prepared liquid
sample to pass from the front end to the back end of the cartridge.
At an appropriate point in the test, after the liquid sample has
been prepared and is within the back end of the cartridge for
analysis, the isolation valve 50 is closed to prevent any of the
sample from re-entering the front end. Once the isolation valve 50
is closed, it cannot be opened again. The isolation valve 50 also
acts as a safeguard in case of a power failure, wherein the reader
closes the isolation valve 50 to prevent leakage.
[0125] Downstream of the isolation valve 50, the fluid pathway
splits into an amplification test channel 54a and an amplification
control channel 54b. Each of the amplification channels 54a-b
comprises an amplification chamber 56a-b having an amplification
chamber inlet valve 58a-b and an amplification chamber outlet valve
60a-b. Any nucleic acid amplification method may be performed in
the nucleic acid amplification chamber. If PCR is used, the nucleic
acid amplification chambers contain a thermostable DNA polymerase,
dNTPs, a pair of primers which are capable of hybridising to the
nucleic acid to be amplified. Optionally, the nucleic acid
amplification chambers may additionally contain buffer salts,
MgCl.sub.2, passivation agents, uracil N-glycosylase and dUTP. An
example of a thermostable DNA polymerase that may be used is Taq
polymerase from Thermus aquaticus.
[0126] Each of the nucleic acid amplification chambers in the
exemplary cartridge comprises reagent containment features in the
form of first and second shallow wells formed in the fluidic layer.
The reagents to be used in the cartridge are spotted in the wells.
In the exemplary cartridge, the test-specific reagents and the
generic reagents are isolated from each other by spotting each in a
different well. Hence, the test-specific reagents are spotted in a
first well in the chamber and the generic reagents are spotted in a
second well in the chamber. By spotting the reagents separately, it
is easier to swap the test-specific reagents during manufacture for
a different set of test-specific reagents, so as to perform a
different test, whilst keeping the generic reagents as they
are.
[0127] In the exemplary cartridge, the ratio of nucleic acid
amplification chambers to detection chambers is 1:2. The prepared
sample enters the back end of the cartridge at the isolation valve
50 and is split into two nucleic acid amplification chambers. After
processing, the each of the two processed measures of sample from
the nucleic acid amplification chamber is split into two detection
chambers. Therefore, for each sample introduced into the exemplary
cartridge, four detection chambers may be filled from two nucleic
acid amplification chambers, thus facilitating duplex amplification
and 4-plex detection.
[0128] However, it will be appreciated that one or three or more
nucleic acid amplification chambers may be provided to provide any
level of multiplexing desired, and that the number of the detection
chambers provided may be adjusted accordingly to maintain a 1:2
ratio of nucleic acid amplification chambers to detection
chambers.
[0129] The ratio 1:2 is preferred for the exemplary cartridge
because such a ratio allows twice the number of target nucleic
acids to be assayed compared to the number of different labels
required for detection in the detection chambers. However, it will
be appreciated that the ratio may be changed depending on the
number of labels and PCR targets for the liquid sample. For
instance, the ratio may be 1:1, 1:3 or 1:n such that there are n
detection chambers branching from the main channel of each fluid
pathway when there are n times as many multiplexed PCR targets for
the number of labels.
[0130] PCR primers specific for Chlamydia trachomatis are dried
down in the amplification chamber in the amplification test channel
together with the other reagents required for nucleic acid
amplification. PCR primers specific for a positive control nucleic
acid are dried down in the amplification chamber in the
amplification control channel together with the other reagents
required for nucleic acid amplification. A positive control nucleic
acid is also provided in the amplification chamber in the
amplification control channel, taken from Pectobacterium
atrosepticum. The dried down reagents are reconstituted when the
liquid sample reaches them.
[0131] Downstream of the amplification chamber outlet valves 60a-b
each of the amplification channels 54a-b splits into two further
detection channels, leading to two detection chambers for each
amplification chamber, giving a total of four detection chambers
62a-d in total. The reagents for nucleic acid detection, including
the target probe, are dried down in the detection chambers 62a-d
downstream of the test amplification chamber 55a or 56b. The
reagents for nucleic acid detection including the control probe are
dried down in the detection chambers downstream of the control
amplification chamber 56a or 56b (whichever is not the test chamber
mentioned above). Each detection chamber 62a-d is provided with its
own gas spring 64a-d which forms a dead end at the downstream end
of the fluid pathway.
[0132] Reagents for nucleic acid detection are provided in
detection chambers. The reagents for nucleic acid detection include
probes having a ferrocene label. These probes are capable of
hybridising to the amplified nucleic acids. Following hybridisation
of the probes to the amplified nucleic acids, the probes are
hydrolysed by a double strand specific nuclease which causes the
label to be freed from the rest of the probe. As explained above,
freeing of the label from the rest of the probe causes a detectable
change in the signal from the label. The control probe is provided
in separate detection chambers to the target probe and detection of
the target nucleic acid and the control nucleic acid take place in
different detection chambers, such that the signals are
distinguishable from one another.
[0133] Downstream of the amplification outlet valves 60a-b, but
upstream of the forks creating the four detection channels, two
bypass channels 66a-b respectively join the two amplification
channels 54a-b. The purpose of the bypass channels 66a-b is to
remove excess liquid sample within the amplification channels 54a-b
before the liquid sample enters the detection chambers 62a-d. The
bypass channels 66a-b connect to a bypass valve 68, which is also
fluidly coupled to the elution chamber branch 16b of the main
channel 16, downstream of the isolation valve 50, before the
channel splits into amplification channels 54a and 54b.
[0134] A particular arrangement of a preferred chamber in the
cartridge, such as the first and second amplification chambers or
the first to fourth detection chambers, may itself form an isolated
inventive aspect of the cartridge, as described further in section
3, below.
[0135] It will be appreciated that the number of amplification
chambers, and the number of detection chambers in the exemplary
cartridge may vary depending on the preferred implementation.
Moreover, other configurations of channels, chambers, valves and so
on are possible without departing from the scope of the invention,
as defined by the claims.
[0136] The physical structure and operation of the various
components of the exemplary cartridge introduced above will now be
explained with reference to FIGS. 2 to 10.
[0137] 1.3 Physical Structure of an Exemplary Cartridge
[0138] 1.3.1 Overview and External Features of the Exemplary
Cartridge
[0139] An exemplary cartridge is shown in FIG. 2. As described
above, the reader interacts with the cartridge through a plurality
of interfaces. The interfaces shown in the exemplary cartridge 100
are: a pneumatic interface 101; an electrical interface 102; a
bypass valve interface 103; and an isolation valve interface 104.
Each of these interfaces is described in more detail below. It will
be appreciated that more or fewer interfaces could be provided,
depending on the preferred implementation.
[0140] Also provided in the cartridge, but not shown, is a thermal
interface. The thermal interface allows the temperature of the
amplification chambers to be regulated to allow nucleic acid
amplification to take place.
[0141] The exemplary cartridge 100 shown in FIG. 2 comprises an
insertion end 105 for insertion into the reader, and a
non-insertion end 106. Proximate the non-insertion end 106 is a
sample inlet 107 for introducing a sample into the sample mixing
chamber 10. In the exemplary cartridge, the sample will usually
include cells, and the target nucleic acid (if present) can be
extracted from these cells, but other fluid samples such as swab
eluate, urine, semen, blood, saliva, stool sweat and tears could be
used in other implementations. The sample may be introduced into
the sample mixing chamber 10 through the sample inlet 107 using a
pipette, for example.
[0142] The exemplary cartridge 100 and reader are configured such
that when the cartridge is inserted into the reader, all of the
aforementioned interfaces are actuatable by the reader. On the
other hand, the sample inlet 107 remains external to the reader
such that a sample may be introduced into the sample mixing chamber
10 whilst the cartridge is inserted into the reader.
[0143] The exemplary cartridge 100 shown in FIG. 2 further
comprises a sample indicator window 109, through which the sample
indicator 12 is visible to determine whether a sample has been
introduced into the sample mixing chamber 10.
[0144] All of the pneumatic, mechanical and electrical interfaces
in the exemplary cartridge 100 are located on the same face of the
cartridge, in this case the top face 110. The thermal interface
(not shown) is provided on the bottom face of the cartridge. This
simplifies the design of the reader, which may this provide the
associated pneumatic, mechanical and electrical parts which
interact with those interfaces in the same region of the reader,
thereby making best use of space. It also enables the thermal part
of the reader to be provided away from the pneumatic, mechanical
and electrical parts.
[0145] 1.3.2 Internal Components of Cartridge
[0146] The exemplary cartridge 100 shown in FIG. 2 is formed from
various components which shall now be described. FIG. 3 shows an
exploded view of the exemplary cartridge 100 of FIG. 2. The
cartridge 100 comprises, from top to bottom, a housing 111, a
blister sub-assembly 112, a pneumatic foil 113, a pneumatic layer
114, a fluid layer 115 and a fluidic foil 116. Also shown in FIG. 3
is an electrode layer 117, two filters 118 and a plurality of
absorbent pads 119, which will be described in more detail
below.
[0147] The housing 111 is manufactured from acrylonitrile butadiene
styrene. The pneumatic and fluidic foils 113, 116 are manufactured
from a polyethylene terephthalate/polypropylene composite. The
pneumatic and fluidic layers 114, 115 are manufacture from
polypropylene.
[0148] With the exception of the housing 111, filters 118 and pads
119, each of the components mentioned in the previous paragraph is
adhered to its adjacent component or components. Hence, the blister
sub-assembly 112 is adhered to the pneumatic foil 113, which is
adhered to the pneumatic layer 114, which is adhered to the fluidic
layer 115, which is adhered to the fluidic foil 116. The electrode
layer 117 is adhered to fluidic layer 115 also.
[0149] The adhesion of the layers to each other provides a series
of fluid-tight channels in the cartridge, together with associated
chambers, valves, pumps, bellows and other components. The channels
passing a liquid sample therethrough are liquid-tight and the
channels passing a gas therethrough are gas-tight. Optionally, all
components are both liquid tight and gas-tight. For example,
recesses and openings formed in one or both sides of the pneumatic
and fluidic layers create, when sandwiched together and adhered to
the pneumatic and fluidic foils, respectively, the shapes necessary
to provide the aforesaid channels, chambers, valves, pumps, bellows
and other components.
[0150] Each of the components referred to above in FIG. 3 will now
be described in more detail.
[0151] 1.3.3 Housing 111
[0152] FIG. 4 shows housing 111 in more detail. As shown, housing
111 comprises a generally rectangular upper surface 120 and walls
121 depending therefrom on all four sides (two of which are visible
in FIG. 4). A principal purpose of the housing 111 is to protect
certain components of the cartridge, most notably the blister
sub-assembly 112 and the isolation valve interface 104. It will
therefore be noted that the housing 111 is shorter than the
pneumatic and fluidic layers 114, 115 such that it overlies only a
portion of those layers when the cartridge 100 is assembled. In the
exemplary cartridge 100, the pneumatic interface 101, electronic
interface 102, and bypass valve interface 103 are not covered by
the housing 111 to provide ease of access by the reader.
[0153] The upper surface 120 of the housing 111 has three apertures
122a-c therein, each having walls depending from the peripheries of
the apertures to form, when the cartridge is assembled, three
recesses. The purpose of the recesses is to house the blisters of
the blister sub-assembly 112 such that the blisters may be accessed
and pressed by the reader, but are otherwise protected from
accidental impact. Naturally, since the exemplary cartridge
comprises three blisters, the housing 111 comprises three
corresponding apertures 122a-c forming three corresponding
recesses. It will be appreciated that more or fewer blisters,
apertures and recesses may be provided, depending on the preferred
implementation. Alternatively, the housing 111 could comprise a
single aperture forming a single recess housing all available
blisters.
[0154] The side walls 121 of the housing 111 which run along the
length of the housing 111 between the insertion end 105 and the
non-insertion end 106 of the cartridge 100 comprise flanges 123
along at least a portion of their lower edges. The purpose of the
flanges 123 is two-fold. Firstly, they comprise one or more windows
124a-b for receiving a corresponding number of tabs formed in the
pneumatic layer 114 to hold the cartridge 100 together. Secondly,
the flanges 123 are dimensioned so as to protrude beyond the lower
surface of the fluidic foil 116 when the cartridge is assembled,
such that the fluidic foil 116 is suspended above a flat surface on
which the cartridge 100 is placed. This prevents accidental damage
to the fluidic foil 116 which could otherwise result.
[0155] Although in the exemplary cartridge depicted in FIG. 4
flanges 123 are provided along substantially the length of two
opposing sides of the cartridge, it will be appreciated that
flanges may be provided along three or four edges of the cartridge
and still suspend the foil above a flat surface on which the
cartridge is placed. Similarly, although the cartridge depicted in
FIG. 4 shows flanges 123 extending along substantially the entire
length of the edge, a flange which extends only partially along an
edge may be provided, or multiple flanges may be provided along
each edge.
[0156] The housing 111 further comprises, at the non-insertion end
106, a grip 125 to facilitate insertion of the cartridge into and
removal of the cartridge 100 from the reader by hand. The grip 125
comprises a series of ridges and grooves formed in the housing 111,
but alternative structures to increase friction, such as knurls,
are also possible.
[0157] The housing 111 further comprises a sample inlet aperture
126 through which a sample may be introduced into the sample mixing
chamber 10 of the cartridge 100 using a pipette, for example.
Surrounding the inlet aperture 126 for a given diameter is a basin
127 recessed into the upper surface 120 of the housing 111 to
accommodate a certain amount of spillage of the liquid sample.
Whilst the basin 127 of the exemplary embodiment is substantially
flat, it may be sloped toward the inlet aperture 126, such that any
spillage drains through the inlet aperture 126.
[0158] The exemplary housing 111 further comprises a plurality of
cut-outs: a first cut-out 128 forming the sample window 109, and a
second cut-out 129 to provide access to the isolation valve
interface 104. As with the recesses which protect the blisters, by
providing access to the isolation valve interface 104 only through
a cut-out 129 in the housing 111, the isolation valve interface 104
is protected to some extent from accidental impact, which could
actuate the isolation valve and render the cartridge
inoperable.
[0159] 1.3.4 Blister Sub-Assembly 112
[0160] FIG. 5 shows the blister sub-assembly 112 in more detail.
The blister sub-assembly 112 may be manufactured separately, during
which the blisters are pre-filled with the liquid reagents
necessary for conducting the preferred test, and subsequently
adhered to the pneumatic foil 113.
[0161] Blister sub-assemblies (or `blister packs`) are familiar to
a skilled person. A blister is a collapsible chamber for containing
a liquid, which may be expelled from the blister by pressing on the
blister and thereby collapsing it. In typical blister packs, the
chamber of a blister is sealed by a foil or other frangible layer
which ruptures once the pressure inside the chamber reaches a
particular magnitude as the blister is collapsed.
[0162] In the exemplary cartridge, the blister sub-assembly 112
comprises three blisters 130a-c. These contain, respectively, the
lysis buffer which comprises reagents capable of performing cell
lysis, the wash buffer and the elution buffer.
[0163] The exemplary blister sub-assembly 112 comprises a substrate
131 onto which the aforementioned blisters 130a-c are formed by a
deformable polymeric layer which is shaped to provide the chambers.
Three apertures 132a-c, corresponding to the three blisters 130a-c,
pass through the substrate 132. Each of the apertures is covered by
the deformable polymeric layer forming the chamber, which thereby
connects the aperture to the chamber but for a seal 133a-c between
the respective apertures 132a-c and chambers. Upon application of a
suitable pressure on the blister 130a-c, the seal 133a-c breaks,
thereby causing the liquid contents of the blister to be ejected
from the blister and to flow through the aperture 132a-c in the
substrate 131 out of the blister sub-assembly.
[0164] As shown, the seals 133a-c at least partially surround the
periphery of the chambers, where they meet the substrate 131. At
the point in each seal 133a-c which is designed to break (thereby
forming the liquid passageway between the aperture 132a-c and
chamber), the seal 133a-c may be weaker than the rest of the
periphery. This ensures that the correct part of the seal 133a-c
breaks when the suitable pressure is applied.
[0165] The blisters may be collapsed by the reader when the
cartridge is inserted therein. One or more mechanical actuators
(such as a foot) may be applied by the reader into the recess so as
to collapse the blister.
[0166] The blister sub-assembly 112 further comprises two reference
holes 134a-b configured to permit an assembly fixture to provide a
reference to facilitate positioning of the assembly during
manufacture.
[0167] 1.3.5 Pneumatic Layer 114
[0168] FIGS. 6A and 6B show the pneumatic layer 114 in more detail.
FIG. 6A is a top view of the pneumatic layer and FIG. 6B is a
bottom view. The pneumatic layer 114 is comprised of a rigid
plastic layer 135 which, in certain places, is overmoulded with a
plurality of flexible membranes to form certain components when the
cartridge is assembled. The flexible membranes are manufactured
from a thermoplastic elastomer.
[0169] The rigid plastic layer 135 has a plurality of
differently-shaped recesses therein and apertures therethrough. In
combination with the fluidic layer 115, certain recesses within,
and/or apertures through, the rigid plastic layer 135 form a number
of components, including: the sample mixing chamber 136; the waste
chamber 137; the capture column 138; the elution chamber 139; the
first and second amplification chambers 140a-b; and the first to
fourth detection chambers 141a-d. An aperture 142 is also provided
to give access to the electrode layer 117.
[0170] In combination with the overmoulded flexible membranes and
the pneumatic foil 113, certain other apertures through the rigid
plastic layer form a number of other components, including: the
upstream bellows valve 142; the bellows 143; a pneumatic interface
144; the downstream bellows valve 145; the wash buffer inlet valve
146; the wash buffer air inlet valve 146a; the elution buffer inlet
valve 147; the elution buffer air inlet valve 147a; the waste
chamber valve 148; the elution chamber valve 149; the isolation
valve 150; the first and second amplification chamber inlet valves
151a-b; and first and second amplification chamber outlet valves
152a-b. A further aperture, in combination with an overmoulded
flexible membrane (but not the pneumatic foil) forms a bypass valve
153.
[0171] With the exception of the isolation valve 150 and the bypass
valve 153, the valves formed in the pneumatic layer are
pneumatically-operable valves. That is, each valve is operable to
open and close a fluidic channel in which the valve is located, and
this valve is actuated by applying a particular pressure to a
pneumatic control line coupled to the valve. The pneumatic control
lines are coupled to the pneumatic interface 144, to which the
reader has access when the cartridge 100 is inserted therein.
Hence, to actuate a given pneumatic valve, the reader merely
applies an appropriate pressure to the pneumatic control line
associated with that valve to open or close the valve.
[0172] The isolation valve 150 and the bypass valve 153 are also
actuated by the reader, but mechanically. Again, each valve is
operable to open and close a fluidic channel in which the valve is
located, but the valve is actuated by applying one or more
mechanical actuators (such as a foot) to the valve.
[0173] The pneumatic layer further comprises two reference holes
154a-b configured to permit an assembly fixture to provide a
reference to facilitate positioning of the layer during
manufacture. When the cartridge is assembled, the reference holes
154a-b in the pneumatic layer align with the reference holes 134a-b
in the blister sub-assembly.
[0174] The pneumatic layer further comprises apertures 155a-c
which, when the cartridge is assembled, line up with apertures
132a-c passing through the substrate 131 of the blister
sub-assembly (through the pneumatic foil, as described below).
[0175] 1.3.6 Pneumatic Foil 113
[0176] FIG. 7 shows the pneumatic foil 113 in more detail. As
explained above, the pneumatic foil 113 is adhered to the upper
surface of the pneumatic layer 114, thereby fluidly sealing
channels, chambers, valves, pumps, bellows and other components
formed therein. Thus, for the most part, the pneumatic foil 113 is
a generally rectangular and planar foil sheet so as to provide an
effective seal. Beneficially, the pneumatic foil 113 is inert such
that is does not react with the reagents which move through the
pneumatic layer 114.
[0177] However, the pneumatic foil 113 does not overlie the entire
pneumatic layer 114. In particular, the pneumatic foil 113 does not
overlie the sample mixing chamber 136 or the waste chamber 137 at
the non-insertion end 106 of the cartridge 100, or the bypass valve
153 at the insertion end 105. Moreover, the pneumatic foil 113
comprises cut-outs 156, 157, such that it does not overlie the
isolation valve 150 or the pneumatic interface 144,
respectively.
[0178] The pneumatic foil 113 further comprises three apertures
158a-c which, when the cartridge 100 is assembled, line up with
apertures 132a-c passing through the substrate 131 of the blister
sub-assembly and 155a-c passing through the pneumatic layer 114.
The apertures 158a-c permit the liquid reagents within the blisters
to pass to the pneumatic layer 114, and thence to the fluidic layer
115 through apertures 155a-c.
[0179] The pneumatic foil 113 comprises two reference holes 159a-b
configured to permit an assembly fixture to provide a reference to
facilitate positioning of the layer during manufacture. When the
cartridge is assembled, the reference holes 159a-b in the pneumatic
foil align with the reference holes in the other layers.
[0180] The pneumatic foil is a composite foil manufactured from a
layer of polyethylene terephthalate, to provide strength, with a
layer of polypropylene on top to provide an inert material for
contacting the liquid sample and buffers, and also to enable the
foil to be heat sealed to the pneumatic layer (also manufactured
from polypropylene.
[0181] 1.3.7 Fluidic Layer 115
[0182] FIGS. 8A and 8B show the fluidic layer 115 in more detail.
FIG. 8A is a top view of the pneumatic layer and FIG. 8B is a
bottom view. The fluidic layer 115 is comprised of a rigid plastic
layer 160. As explained previously, the top side of the fluidic
layer 115 (not shown) is adhered to the bottom side of the
pneumatic layer 113 (see FIG. 5B) such that the various channels,
chambers, valves, pumps, bellows and other components formed by a
combination of the pneumatic and fluidic layers are aligned.
[0183] As with the rigid plastic layer 135 of the pneumatic layer
113, the rigid plastic layer 160 of the fluidic layer 115 has a
plurality of differently-shaped recesses therein and apertures
therethrough. In combination with the pneumatic layer 113 and the
fluidic foil 116, certain recesses within, and/or apertures
through, the rigid plastic layer 160 forms certain components,
including: the sample inlet chamber 136; the capture column 138;
the elution chamber 139; the first and second amplification
chambers 140a-b; and the first to fourth detection chambers 141a-d.
the upstream bellows valve 142; the bellows 143; the pneumatic
interface 144; the downstream bellows valve 145; the wash buffer
inlet valve 146; the wash buffer air inlet valve 146a; the elution
buffer inlet valve 147; the elution buffer air inlet valve 147a;
the waste chamber valve 148; the elution chamber valve 149; the
isolation valve 150; the first and second amplification chamber
inlet valves 151a-b; and first and second amplification chamber
outlet valves 152a-b. An aperture 161 is also provided to give
access to the electrode layer 117.
[0184] Moreover, in combination with the fluidic foil 116 (but not
the pneumatic layer 114), recesses in the fluidic layer 115 also
provides the coarse filter 162, the convoluted mixing channel 163,
and a plurality of channels which, when the cartridge is assembled,
connect the aforementioned components together to enable passage of
the liquid sample and liquid reagents through the cartridge, and
facilitate pneumatic actuation of the valves, pumps, bellows and
other components.
[0185] The fluidic layer comprises two reference holes 164a-b
configured to permit an assembly fixture to provide a reference to
facilitate positioning of the layer during manufacture. When the
cartridge is assembled, the reference holes 164a-b in the fluidic
layer align with the reference holes in the other layers.
[0186] As mentioned above, channels are formed between the
pneumatic interface and the various valve and bellows described
above. In the exemplary cartridge, the pneumatic interface
comprises 11 ports which are connected to the various components as
follows. [0187] Port 1: bellows [0188] Port 2: upstream bellows
valve [0189] first and second amplification chamber inlet valves
[0190] first and second amplification chamber outlet valves [0191]
Port 3: downstream bellows valve [0192] Port 4: wash buffer inlet
valve [0193] Port 5: wash buffer air inlet [0194] Port 6: wash
buffer air inlet valve [0195] elution buffer air inlet valve [0196]
Port 7: elution buffer air inlet [0197] Port 8: elution buffer
inlet valve [0198] Port 9: reference pressure line [0199] Port 10:
elution chamber valve [0200] Port 11: waste chamber valve
[0201] It will be understood that whilst various inventive aspects
of the exemplary cartridge may be implemented using specific ones
of the connections listed above (in particular, the first and
second amplification chamber inlet and outlet valves being
connected to a single port; and the wash and elution buffer air
inlets being connected to a single port); the precise configuration
listed above is not essential.
[0202] 1.3.8 Fluidic Foil
[0203] FIG. 9 shows the fluidic foil 116 in more detail. As
explained above, the fluidic foil 116 is adhered to the lower
surface of the fluidic layer 115, thereby fluidly sealing channels,
chambers, valves, pumps, bellows and other components formed
therein. Thus, for the most part, the fluidic foil 116 is a
generally rectangular and planar foil sheet so as to provide an
effective seal. Beneficially, the foil 116 is inert such that is
does not react with the reagents which move in the pneumatic
layer.
[0204] However, the fluidic foil 116 does not overlie the entire
fluidic layer 115. In particular, the fluidic foil 116 does not
overlie the detection chambers 141a-d at the insertion end 105.
[0205] The fluidic foil 116 comprises two reference holes 165a-b
configured to permit an assembly fixture to provide a reference to
facilitate positioning of the layer during manufacture. When the
cartridge is assembled, the reference holes 165a-b in the fluidic
foil align with the reference holes in the other layers.
[0206] The fluidic foil is a composite foil manufactured from a
layer of polyethylene terephthalate, to provide strength, with a
layer of polypropylene on top to provide an inert material for
contacting the liquid sample and buffers, and also to enable the
foil to be heat sealed to the fluidic layer (also manufactured from
polypropylene.
[0207] 1.3.9 Electrode Layer 117
[0208] Finally, FIG. 10 shows the electrode layer 117 in more
detail. As explained above, the electrode layer 117 is adhered to
the fluidic layer 115. The electrode layer 117 comprises four sets
of detection electrodes 166a-d. Each set of detection electrodes
166a-d comprises first to third electrical contacts 168a-d which
couple with corresponding electrical contacts in the reader when
the cartridge is inserted therein. Preferably, the electrical
contacts are made of silver to optimise the electrical connection.
Preferably electrodes which are silver plated with silver chloride
are used to ensure a the optimal galvanic behaviour.
[0209] Each set of detection electrodes 166a-d comprises a working
electrode 169a-d; a counter electrode 170a-d and a reference
electrode 171a-d. Each of the electrodes is coupled to a respective
electrical contact. Each set of detection electrodes 166a-d also
comprises a dielectric 172a-d covering the interface between the
electrodes and the respective electrical contacts.
[0210] A skilled person understands that electrochemical signalling
may be used to indicate the presence of genetic or immuno targets.
In the exemplary cartridge this process is performed in the first
to fourth detections chambers 141a-d which are optimised to provide
the electrochemical test interface.
[0211] The electrodes 166a-d are arranged such that a liquid sample
within the first to fourth detection chambers 141a-d comes into
contact with the first to fourth sets of electrodes 166a-d. In the
detection chambers, some compounds in the fluid sample (referred to
as the `electrolyte`) have a natural tendency to migrate to
electrodes and swap electrons. This galvanic effect is how
batteries work.
[0212] All combinations of soluble compounds have some
electrochemical activity, and the rate at which this activity
occurs (i.e. the amount of charge exchanged) is determined by
exactly what those compounds are. Hence, it is possible to measure
the presence of different analytes in the liquid sample, by
searching for characteristic features of their redox
electrochemistry.
[0213] In the exemplary cartridge, the current required to maintain
a given redox state in the detection chambers 141a-d is monitored
at different redox states. Current is supplied through the
electrolyte from the working electrodes 169a-d to counter
electrodes 170a-d.
[0214] The reference electrodes 171a-d also contact the
electrolyte. Voltages are declared with respect to this reference
electrode because its voltage is largely independent of the redox
conditions and this therefore means that it is only the redox state
of the chemistry at the control electrode that is being
measured.
[0215] A voltage sweep is applied between the working electrodes
169a-d and counter electrodes 170a-d by the reader, which generates
the characteristic range of redox conditions. The current passing
between the working electrodes 169a-d and the counter electrodes
170a-d is then measured to obtain the test results. The voltage
sweep is a slowly incrementing set of voltages applied between the
electrodes. Preferably the sweep is from about -0.7 volts to about
+1 volts relative to the reference electrode. The voltage is
applied in consecutive incrementing pulses having a pulse
modulation amplitude of between 30 and 80 millivolts (preferably
between 40 and 60 millivolts; more preferably 50 millivolts).
Preferably the step increment from one pulse to the next is between
1 and 5 millivolts (preferably between 2 and 4 millivolts; more
preferably 3 millivolts). By applying these voltages across the
electrodes, current measurements in the scale of 100s of nano amps
may be obtained.
[0216] The particular arrangement of detection electrodes
illustrated in FIG. 10 may itself form an isolated inventive aspect
of the cartridge. Conventionally, the counter electrode in a
potentiostat is larger than the working electrode to provide an
ample supply of surplus electrons. However, it has been found that
reversing this convention surprisingly offers better results for
the exemplary cartridge. For the electrochemistry performed on the
liquid sample described above in the exemplary cartridge, it is
found that having a working electrode which is larger than the
counter electrode provides larger signals and improved results by
way of increased sensitivity. In other words, having a current flow
from a relatively large working electrode to a relatively small
counter electrode offers improvements over the conventional
arrangement.
[0217] Preferably each working electrodes 169a-d is formed in a
U-shape and each counter electrode 170a-d is formed in a straight
elongate shape between the two prongs of the respective U-shaped
working electrode.
[0218] The method operation of the exemplary cartridge introduced
above will now be briefly explained.
[0219] 1.4 Method of Operation of the Exemplary Cartridge
[0220] 1.4.1 the Front End
[0221] As described above, a fluid sample (such as a urine sample)
is introduced into the sample mixing chamber 10 using a pipette. A
portion of the sample passes to the sample indicator 12 to show
that a sample is present in the sample mixing chamber.
[0222] Once the cartridge 100 with a sample in the mixing chamber
10 is inserted into a reader, and the reader is activated, the test
may commence. Firstly, the reader will apply a mechanical actuator
(such as a foot) to collapse the lysis buffer blister 14. In doing
so, the lysis buffer will be expelled into the sample mixing
chamber 10 where it will mix with the sample.
[0223] The bellows 20 and its valves 22a-b then moves the liquid
sample and lysis buffer back and forth into the sample mixing
chamber 10 so as to mix the lysis and sample and to rehydrate the
internal control. Following the mixing step, incubation of the
sample and lysis buffer occurs to allow cell lysis to take
place.
[0224] The bellows 20 and its valves 22a-b will then commence
operation to pump the sample from the sample mixing chamber 10,
into the main channel 16, through the coarse filter 18 and toward
the capture column 24. Within the capture column 24 nucleic acids
are specifically bound to a filter in the capture column on the
basis of their size and charge. The unwanted liquid sample passes
through to the waste chamber 38.
[0225] Once the unwanted liquid sample has passed to the waste
chamber 38, leaving the nucleic acids bound to the capture column
24, the reader applies a mechanical actuator (such as a foot) to
collapse the wash buffer blister 30. In doing so, the wash buffer
will be expelled into the first branch channel 26, and thence into
the main channel 16. Again, the bellows 20 and its valves 22a-b
will commence operation to pump the wash buffer through the main
channel 16 and through the capture column 24 to wash any remaining
unwanted cell debris and other cellular components out of the
capture column with the wash buffer through to the waste chamber
38, or else the wash buffer will be flushed into the waste chambers
using air from the wash and/or elution buffer air inlets.
[0226] Once the wash sample has passed to the waste chamber 38,
leaving only the bound and purified nucleic acids in the capture
column 24, the reader applies a mechanical actuator (such as a
foot) to collapse the elution buffer blister 32. In doing so, the
elution buffer will be expelled into the second branch channel 28,
and thence into the main channel 16. Again, the bellows 20 and its
valves 22a-b will commence operation to pump the elution buffer
through the main channel 16 and through the capture column 24 to
elute the nucleic acids from the capture column, or else the
elution buffer will be flushed into the capture column using air
from the wash and/or elution buffer air inlets. The prepared liquid
sample then passes through to the elution chamber 46; again, either
by being pumped or flushed as described above
[0227] The sample settles in the elution chamber 46 allowing
bubbles to disperse before entering the amplification chambers.
[0228] 1.4.2 the Back End
[0229] The bellows 20 and its valves 22a-b will then commence
operation to pump the liquid sample from the elution chamber 46,
through the isolation valve 59, through the mixing channel 52 and
into the amplification chambers 56a-b, or else the sample will be
flushed into the amplification chambers using air from the wash
and/or elution buffer air inlets. In the nucleic acid amplification
chambers 56a-d the nucleic acid of interest, if present, is
amplified such that it Is present at a detectable level. The
control nucleic acid is also amplified such that it is present at a
detectable level. As mentioned above, any nucleic acid
amplification method may be used. Where PCR is used, primers
specifically hybridise to the nucleic acid of interest and are
extended by a thermostable polymerase such as Taq polymerase via
the addition of dNTPs to the 3' end of each of the primers. Any
excess liquid sample may be removed from the fluid pathway through
the bypass channels 68.
[0230] The bellows 20 and its valves 22a-b will then commence
operation to pump the liquid sample from the amplification chambers
56a-b and into the detection chambers 62a-d, or else the sample
will be flushed into the detection chambers using air from the wash
and/or elution buffer air inlets. In the detection chambers, the
target probe specifically hybridises to the target amplified
nucleic acid of interest and the control probe specifically
hybridises to the amplified control nucleic acid. The nuclease
hydrolyses the target and control probes following hybridisation of
the probes to the amplified nucleic acid. The hydrolysis of the
target and control probes frees the labels from the probes causing
a detectable change in the signal from the labels to occur.
[0231] Once the liquid sample occupies the detection chambers, the
reader applies a mechanical actuator to the isolation valve 50 to
close the valve and isolate the liquid sample in the back end of
the device.
[0232] The electrodes provide a potential difference across the at
least one detection chamber. Depending on the state of the label
(i.e. whether it is attached to the full length probe or the probe
has been hydrolysed and it is free or attached to a single
nucleotide or short part of the probe), the current that is able to
flow through the detection chamber will differ. The electrodes
therefore allow detection by the reader of the change in the signal
from the label which results from hydrolysis of hybridised
probe.
[0233] The present invention will now be described with reference
to FIGS. 18 to 25.
[0234] 2. Channels and Method for Clearing Dead-Legs
[0235] The following section describes the present invention in
more detail with reference to FIGS. 18 to 25. The invention may be
implemented in the fluidic cartridge described above, specifically
in the portion of the main channel between the bellows 20 and the
capture column 24, wherein a plurality of fluids are introduced
from branch channels into the main channel. The invention may be
practiced with a main channel and just a single branch channel, but
any number of branch channels may be provided, depending on the
preferred implementation.
[0236] FIG. 18a illustrates a first embodiment of the invention,
comprising main channel B100 and a first branch channel B101. The
main channel B100 comprises an upstream end B100a and a downstream
end B100b, wherein the upstream end B100a is capable of receiving a
sample and passing the liquid sample from the upstream end B100a to
the downstream end B100b. The branch channel B101 joins the main
channel B100 in the region of the main channel B100 between these
two ends.
[0237] The first branch channel B101 further comprises a gas inlet
B101a for introducing a gas into the first branch channel, a liquid
inlet B101d for introducing a liquid into the first branch channel,
and a valve B101c. The valve B101c is configured to move between a
closed position in which it prevents liquid and gas in the first
branch channel from passing into the main channel and an open
position in which it permits liquid and gas in the first branch
channel to pass into the main channel.
[0238] FIG. 18b illustrates an alternative arrangement of the first
embodiment of the invention, wherein the first branch channel
further comprises a gas inlet valve B101b to prevent liquid and gas
flowing from the first branch channel through the gas inlet B101a.
The gas inlet valve B101b is configured to move between a closed
position in which it prevents liquid in the first branch channel
from passing into the gas inlet and an open position in which it
permits gas from the gas inlet to pass into the first branch
channel. The gas inlet valve reduces the risk of contamination of
the gas inlet by the liquid from the liquid inlet. Optionally, the
gas inlet and gas inlet valve may be coupled together, or in an
alternative embodiment the gas inlet and gas inlet valve may be
independent.
[0239] FIG. 19a illustrates a second embodiment of the invention,
wherein the fluidic cartridge includes a main channel B100, a first
branch channel B101 and a second branch channel B102. The main
channel B100 comprises an upstream end B100a and a downstream end
B100b, wherein the upstream end B100a is capable of receiving a
sample and passing the liquid sample from the upstream end B100a to
the downstream end B100b. The first branch channel B101 joins the
main channel B100 in the region of the main channel B100 between
these two ends, and the second branch channel B102 joins the main
channel B100 downstream of the first branch channel B101.
[0240] The second branch channel B102 further comprises a gas inlet
B102a for introducing a gas into the second branch channel, a
liquid inlet B102d for introducing a liquid into the second branch
channel, and a valve B102c. The valve B102c is configured to move
between a closed position in which it prevents liquid and gas in
the second branch channel from passing into the main channel and an
open position in which it permits liquid and gas in the second
branch channel to pass into the main channel.
[0241] FIG. 19b illustrates an alternative arrangement of the
second embodiment, wherein the first and second branch channels
B101, B102 further comprise a gas inlet valve B101b, B102b to
prevent liquid and gas flowing from the branch channel through the
gas inlet B101a, B102a. The gas inlet valve B101b, B102b is
configured to move between a closed position in which it prevents
liquid in the branch channel from passing into the gas inlet and an
open position in which it permits gas from the gas inlet to pass
into the branch channel. The gas inlet valve reduces the risk of
contamination of the gas inlet by the liquid from the liquid inlet.
Optionally, the gas inlet and gas inlet valve may be coupled
together, or in an alternative embodiment the gas inlet and gas
inlet valve may be independent (FIG. 19c). In a further embodiment,
the first and second gas inlet valves B101b, B102b in the first and
second branch channels, respectively, may be actuated
simultaneously as described in more detail below.
[0242] The skilled person would understand that although the
present embodiments illustrate a first and a second branch channel
joining a main channel, any number of additional branch channels
could be fluidically connected to the main channel in any sequence
or orientation.
[0243] The branch channels B101 and B102 in FIGS. 18 and 19 are
illustrated as joining the main channel at a 90-degree junction, or
T-junction. However, the person skilled in the art will appreciate
that the one or more branch channels may join the main channel B100
at any other angle, such as at 90 degrees or an acute angle, all of
which are covered by the scope of this invention.
[0244] The valves B101c, B102c on the one or more branch channels
B101, B102 may be spaced from the main channel B100, thereby
forming a dead-leg B108 in the branch channel B101, B102 between
the valve and the main channel B100 (FIGS. 18 and 19). When the
liquid sample passes along the main channel B100 from the upstream
end B100a to the downstream end B100b, a volume of the liquid
sample will accumulate in the dead-leg B108. Subsequent fluid
passes may be contaminated by the presence of the accumulated
liquid.
[0245] Alternatively, the valves B101c, B102c on the one or more
branch channels B101, B102 may be located at a junction between the
branch channel B101, B102 and the main channel B100. The valve
B101c, B102c may be a T-valve, for instance. Even when such valves
B101c, B102c are provided at the junction, however, it is
impossible to close the valve exactly flush with the main channel
B100 so as to leave the main channel entirely uninterrupted. Hence,
even when the valve B101c, B102c is provided at the junction, there
will always be a section outside of the flow path of the main
channel B100 which will provide a dead-leg B108. When the liquid
sample passes along the main channel B100 from the upstream end
B100a to the downstream end B100b, a volume of the liquid sample
will accumulate in the dead-leg B108.
[0246] In order to clear residual liquid sample from the dead-legs,
it is advantageous to have a gas inlet on the branch channel. The
passage of gas from the gas inlet B101a, B102a on the one or more
branch channels is used to clear the branch channels of residual
liquid sample, and is also be used to dry the branch channels after
the liquid sample has passed through the main channel.
[0247] The liquid inlet B101d, B102d on the one or more branch
channels B101, B102 can be coupled to a liquid chamber. The liquid
chamber may contain liquid reagents or a buffer, where the buffer
can include a lysis buffer, a wash buffer or an elution buffer. In
one embodiment, the liquid chamber coupled to the liquid inlet
B101d in the first branch channel B101 may contain a wash buffer,
and the liquid chamber coupled to the liquid inlet B102d in the
second branch channel B102 may contain an elution buffer. The
introduction of different liquids through independent liquid inlets
B101d on separate branch channels avoids passing different liquids
or reagents through a single inlet, which could lead to cross
contamination of the reagents prior to downstream reactions.
However, the presence of multiple branch channels with individual
liquid inlets B101d, B102d can also lead to the accumulation of
more liquids or reagents in the dead-legs B108 of the branch
channels. The gas inlets B101a, B102a can also be used to clear any
liquids introduced from the liquid inlets B101d, B102d, such as
reagents, from the one or more branch channels B101, B102. It is
particularly beneficial if the gas inlet is located further from
the junction of the branch channel with the main channel than the
liquid inlet. This design allows for complete evacuation of any
residual liquid sample or liquid from the dead-legs B108 in the
branch channel.
[0248] The liquid chamber B500 may be a collapsible blister (such
as those described above) adapted, when it is collapsed, to eject a
liquid contained therein through the liquid inlet B101d, B102d,
into the branch channel B101, B102, and into the main channel B100,
for introducing the liquid into the main channel B100 after the
liquid sample has passed downstream of the one or more branch
channels B101, B102. FIGS. 20a and 20b illustrate a collapsible
blister B500 that may be formed from a polymer material and sealed
with a secondary flat foil B501. The bottom surface of the foil
B501 is secured to an upper surface of the fluidic cartridge B1
using adhesive tape. The seal on the blister, between the foil and
the cartridge, is designed such that it is permanent around the
perimeter of the blister B502, except in a small region B503
adjacent the liquid inlet. In this region B503 the seal is
frangible. On application of a force, for example using a
mechanical actuator, the foils are delaminated in the region of the
frangible seal, where the seal is weakest. The liquid in the
chamber B500 is then released and directed into the liquid inlet
B101d via a fluid channel in the cartridge.
[0249] The liquid chamber fluid capacity can be between 100 and 800
.mu.l, preferably between 200 and 750 .mu.l. The wash buffer
chamber capacity should more preferably be between 400 and 700
.mu.l, and most preferably between 450 and 650 .mu.l. The elution
buffer chamber capacity should more preferably be between 150 and
5000 .mu.l, and most preferably between 200 and 400 .mu.l.
[0250] Many different types of valves for use in controlling fluids
in microfluidic devices have been developed and are contemplated
for use in the present invention. In the exemplary cartridge, the
or each valve or gas inlet valve B101b, B102b in the one or more
branch channels is a pneumatically-actuated valve.
[0251] FIG. 21 illustrates a suitable pneumatically-actuated valve
for use in the present invention. The valve B601d comprises a valve
chamber B600 having a first opening B601a and a second opening
B601b connected to the branch channel B601 The valve B601d further
comprises a flexible membrane B602 movable between a closed
position (FIG. 21a), in which the flexible membrane B602 seals
against the first and second openings B601a, B6501b to prevent
fluid flow through the branch channel B601, and an open position
(FIG. 21b), in which the flexible membrane B602 is spaced apart
from the first and second openings B601a, B601b to permit fluid to
flow through the branch channel. The valve B601c further comprises
a fluid passageway B603 having an opening B603a in the valve
chamber B600. The opening B603a is separated from the first and
second openings B601a, B601b by the flexible membrane B602. The
fluid passageway B603 serves as an actuation channel to move the
flexible membrane between its open and closed positions, and thus
actuates the valve.
[0252] The exemplary fluidic cartridge described above comprises a
pneumatic interface for connecting to a source of positive pressure
and/or gauge pressure. The pneumatic interface comprises a
plurality of ports. The fluid passageway B603 is coupled to a port
in the pneumatic interface for applying a positive pressure and/or
gauge pressure in the valve chamber B600 so as to move the flexible
membrane B602 between the open and closed positions (see FIGS. 21a
and 21b). When a positive gas pressure is applied via the fluid
passageway B603, the pressure within the valve chamber B600
increases beyond that in the branch channel B601 and the flexible
membrane B602 will be forced into the closed position. Conversely,
when a vacuum or gauge pressure is applied via the fluid passageway
B603, the pressure within the valve chamber B600 reduces below that
in the branch channel B601 and the flexible membrane B602 is
brought into the open position.
[0253] The first and second gas inlet valves B101b and B102b in the
first and second branch channels, respectively, may be coupled to
different ports in the pneumatic interface. However, FIG. 22
illustrates a preferred embodiment wherein the first and second gas
inlet valves B101b and B102b in the first and second branch
channels, respectively, are coupled together, to the same port B901
in the pneumatic interface B900, allowing the gas inlet valves
B101b and B102b on the first and second branch channels to be
actuated simultaneously.
[0254] The gas inlets B101a, B102a on the one or more branch
channels B101, B102 may be coupled to a gas supply. Optionally, the
gas inlets B101a, B102a are coupled to the pneumatic interface for
connection to a gas supply. The gas supply is suitable for passing
the gas through the as inlet, into the main channel and can be used
to introduce the gas into the main channel before or after the
liquid sample has passed downstream of the one or more branch
channels B101, B102, preferably after. Optionally, the first and
second gas inlets B101a, B102a in the first and second branch
channels B101, B102, respectively, are coupled to independent ports
on the pneumatic interface for connection to independent gas
supplies. In the exemplary cartridge, the gas supply is provided by
the reader when the cartridge is inserted into the reader.
[0255] The first and second gas inlets B101a, B102a are coupled to
their respective gas supplies via independent gas supply channels
so as to reduce the risk of cross contamination of the gas supply.
In use, the gas is supplied via the gas inlets B101a, B102a and the
gas inlet valves are opened simultaneously via a positive pressure
applied by the pneumatic interface B900 so as to push any liquid
through the branch channels. Provided one or more of the valves
B101c and B102c are open, the gas will evacuate any liquid from the
dead-legs in the one or more branch channels.
[0256] In one embodiment, relating to the presence of two branch
channels, the pneumatic interface B900 may comprise first to third
ports B901, B902, B903. FIG. 23 illustrates the first to third
ports which are respectively coupled to: [0257] i. the valve
chamber of the valve B101c in the first branch channel (coupled to
port B902); [0258] ii. the valve chamber of the valve B102c in the
second branch channel (coupled to port B903); [0259] iii. the valve
chamber of the gas inlet valve in the first branch channel B101b
and the valve chamber of the gas inlet valve in the second branch
channel B102b (coupled to port B901).
[0260] The ports are coupled to the valve chambers so as to actuate
the valves. The valves B101c, B102c on the first and second branch
channels may be operated independently from each other and the gas
inlet valves B101b, B102b or in conjunction with each other so as
to open and close all valves and gas inlet valves simultaneously.
The operation of the valves and gas inlet valves is controlled by
the pneumatic interface B900.
[0261] It should be understood that the terminology "first,
"second" and "third" ports does not necessarily reflect the order
in which the ports are located on the fluidic cartridge or which
the valve chambers are connected to the pneumatic interface.
[0262] The main and branch channels can typically have a diameter
between 0.5 and 1.4 mm, preferably between 0.6 and 1.0 mm and more
preferably between 0.7 mm and 0.9 mm. These diameters are optimised
to permit a high gas flow rate (so as to clear and dry the
channels) and the channel surfaces are smooth to ensure linear
fluid flow.
[0263] The main channel and one or more branch channels may be
formed in the fluidic layer of the cartridge, as described above.
The fluid passageway B603 may be formed in the pneumatic layer of
the cartridge. The fluidic and pneumatic layers may be manufactured
from polypropylene.
[0264] The operation of the fluidic cartridge shown in FIG. 18 will
now be described with reference to the method of processing a
liquid sample B700 of FIG. 24. In operation, the liquid sample can
be passed through the main channel B100. Liquid entering the main
channel flows from the upstream end B100a to the downstream end
B100b. The valve B101c prevents liquid sample flowing from the main
channel B100 and into the branch channel B101. However, liquid
sample is likely to accumulate in the dead-legs B108 of the first
branch channel B101 A gas can be passed through the first branch
channel B101 and into the main channel B100 to remove the
accumulated liquid sample. As such, the gas source is turned on
(step B702) and the gas inlet valve B101b is opened. Gas is then
passed from the gas inlet B101a through the first branch channel
B101 and into the main channel B100 evacuating any residual liquid
sample in the dead-legs B108. Once the dead-leg is cleared the gas
inlet valve B101b can be closed and the gas source turned off.
Closing of the gas inlet valve prevents liquid from passing from
the liquid inlet and into the gas inlet when a liquid is ejected
from the liquid chamber.
[0265] A liquid may then be passed from the liquid inlet B101d
through the first branch channel B101 and into the main channel
B100. The liquid may be ejected from a liquid chamber coupled to
the liquid inlet. The liquid may be a reagent for reacting with the
liquid sample downstream of the main channel. It may be necessary
to precisely control the volume of liquid that reacts with the
liquid sample, and as such it is important that the entire volume
of liquid passes into the main channel B100. Gas can be used to
purge the liquid reagent from the branch channel B101 and the
dead-legs B103 into the main channel B100, thus ensuring the total
volume of liquid reagent is passed to the downstream end B100b of
the main channel. Therefore, the final steps B706 and B707 require
that the gas source is turned on, and the gas inlet valve B101b is
opened so that a gas can be passed from the gas inlet B101a through
the first branch channel B101 and into the man channel B100 so as
to evacuate any residual liquid from the first branch channel
B101.
[0266] The fluidic cartridge may further comprise a gas inlet valve
B101b in the first branch channel (FIG. 18b) such that when the gas
inlet valve is closed, liquid and gas is prevented from flowing
from the first branch channel through the gas inlet B101a. To allow
passage of the gas from the gas supply, through the gas inlet B101a
and into the branch channel, the gas inlet valve B101b should be
opened after the gas supply is turned on. Once the dead-leg is
cleared, the gas inlet valve B101b can be closed and the gas supply
turned off. Closing of the gas inlet valve prevents liquid from
passing from the liquid inlet and into the gas inlet when a liquid
is ejected from the liquid chamber.
[0267] The gas should be supplied to the gas inlet B101a be prior
to opening the gas inlet valve B101b or valve B101c on the first
branch channel so as to maintain a forward pressure out of the air
inlet B101a
[0268] The operation of the fluidic cartridge shown in 19b will now
be described with reference to the method of processing a liquid
sample B800 of FIG. 20. In operation, the liquid sample can be
passed through the main channel B100. Liquid entering the main
channel flows from the upstream end B100a to the downstream end
B100b. The valves B101c, B102c prevent liquid sample flowing from
the main channel B100 and into the first or second branch channels
B101, B102. However, liquid sample is likely to accumulate in the
dead-legs B108 of the first and second branch channels B101, B102.
To evacuate any accumulated liquid sample and also to purge the
liquid sample from the main channel, a gas can be passed through
the first and second branch channels B101, B102 and into the main
channel B100. As such, gas is supplied to the first and second gas
inlets B101a, B102a (step B802), the gas inlet valves B101b, B102b
are opened and the valves B101c, B102c are also opened. If the gas
inlet valves are both connected to the same port on the pneumatic
interface B900 they will be opened simultaneously. Gas is then
passed from the gas inlet B101a, B102a through the first and second
branch channels B101, B102 and into the main channel B100
evacuating any residual liquid sample in the dead-legs B108. Once
the dead-legs are cleared the gas inlet valves B101b, B102b can be
closed and the gas source turned off. Closing of the gas inlet
valve prevents liquid from passing from the liquid inlets and into
the gas inlet when a liquid is ejected from the liquid chamber. To
ensure the pressure residues in the first and second branch
channels are released to atmospheric pressure after the gas has
cleared the branch channels, the upstream and downstream bellows
pump valves 22a-b can be temporarily opened.
[0269] The valves on the first and second branch channels can then
be opened or closed to allow for the passage of liquid from the
liquid inlet on one of the branch channels. Preferably the liquid
from the liquid inlet on the first branch channel B101d is passed
through the channels before the liquid from the liquid inlet on the
second branch channel B102d. This reduces the risk of
cross-contamination of the second liquid reagent with the first
liquid reagent by reducing the fluid path length of the second
liquid reagent. To further avoid cross-contamination of the branch
channels the valves can be used to restrict the flow of the liquid.
In particular, closing the valve B102c on the second branch channel
before passing a liquid from the liquid inlet on the first branch
channel B101d through the first branch channel B101 and into the
main channel B100 prevents liquid from the first branch channel
flowing any further into the second branch channel than the
dead-leg B108.
[0270] In a particular use, the liquid from the liquid inlet on the
first branch channel may be a wash buffer. Since wash buffers can
often be toxic to downstream reactions it is important to ensure
the wash buffer is removed before any further liquid passes through
the channels. Gas can be used to evacuate any dead-legs B108 and
also to ensure the first and second branch channels and the main
channel are clear before any further liquid passes. The next step
B807 of the method B800 includes turning the gas source connect to
the gas inlet B101a on the first branch channel on, and turning the
gas source connect to the gas inlet B102a on the second branch
channel on. The valve B102c on the second branch channel and the
gas inlet valves B101b, B102b on the first and second branch
channels are then opened and a gas is passed from the gas inlet
B101a through the first branch channel B101 and from the gas inlet
B102a through the second branch channel B102 into the main channel
B100. Opening both of the valves B101c, B102c ensures both
dead-legs in the first and second branch channels are cleared of
any residual liquid. Once the dead-legs are cleared the gas inlet
valves B101b, B102b can be closed and the gas sources turned off.
To ensure the pressure residues in the first and second branch
channels are released to atmospheric pressure after the gas has
cleared the branch channels, an upstream and a downstream bellows
pump valve can be temporarily opened.
[0271] A second liquid (which may or may not be the same as the
first liquid) may then be passed from the liquid inlet B102d
through the second branch channel B102 and into the main channel
B100. The valve B101c on the first branch channel is closed to
prevent any back flow of the liquid into the first branch channel,
and the valve B102c on the second branch channel is opened. The
liquid is passed from the liquid inlet on the second branch channel
B102d through the second branch channel B102 and into the main
channel B100. In a particular use, the liquid from the liquid inlet
on the second branch channel may be an elution buffer. Subsequent
gas passes are used to ensure that the total volume of elution
buffer has passed through the branch channels and into the main
channel to react with the liquid sample. The final steps B811 and
B812 require that gas is supplied to the gas inlets B101a, B102a on
the first and second branch channels so that a gas can be passed
from the gas inlets B101a, B102a through the first and second
branch channels and into the main channel B100. The total volume of
elution buffer should thus have passed into the downstream end of
the main channel B100b.
[0272] When passing the gas through the branch channels and main
channel, gas is supplied to the gas inlets B101a, B102a prior to
opening the gas inlet valves B101b, B102b or valve B101c, B102c on
the first and second branch channels. This maintains a forward
pressure out of the air inlets B101a, B102a. The one or more steps
of passing a gas from the gas inlet B101a, B102a on the one or more
branch channels and into the main channel B100 further comprises
purging the residual liquid sample and/or residual liquid from the
B100 main channel. The liquid sample and/or residual liquid may
pass into a receptacle chamber such as a reaction chamber, capture
column or waste chamber.
[0273] The gas inlet valves B101b, B102b are provided so that when
they are closed they prevent any liquid from passing into the gas
inlets B101a, B102a.
[0274] The fluidic cartridges may further comprise a pneumatic
interface, and it is this interface which controls the actuation of
the valves B101c, B102c and gas inlet valves B101b, B102b. The gas
supplies may also be connected to ports on the pneumatic interface
which can be coupled to the gas inlets B101a, B102a. The pneumatic
interface can be used to supply a source of positive gas pressure
which is then passed through the gas inlets B101a, B102a and into
the branch channels B101, B102.
[0275] The steps of evacuating any residual liquid from the branch
channels results in the cleaning of the dead-legs such that no
residual liquid (be that liquid sample, or liquid from the liquid
inlets) remains within the branch channels. This reduces the risk
of later contamination of the reagents or sample. As described the
presence of any wash buffer may be `toxic` to the subsequent
downstream reactions, particularly in sensitive reactions such as
PCR. Elution buffers are prevented from acting if any trace wash
buffer remains, and thus it is important to ensure all wash buffer
is removed before passing the elution buffer through the
channels.
[0276] The steps of passing a liquid from the liquid inlet may
comprise expelling the liquid from the liquid chamber into the
branch channel. Where the liquid chamber is a collapsible blister,
the step of passing a liquid from the liquid inlet comprises
collapsing the collapsible blister and thereby ejecting liquid
contents through the liquid inlet, into the branch channel and into
the main channel.
[0277] It should be recognized that other arrangements,
configurations and methods should be readily apparent to a person
of ordinary skill in the art. Further branch channels can be joined
to the main channel, and as such, various reagents can be contained
within the liquid chambers connected to the branch channel liquid
inlets. Furthermore, any pneumatically-actuated valve can be
replaced by any other suitable microfluidic valve. It should be
understood that various alternatives to the embodiments of the
invention described herein can be employed in practicing the
invention. It is intended that the following claims define the
scope of the invention and that methods and structures within the
scope of these claims and their equivalents be covered thereby.
[0278] 3. Additional Isolated Inventive Aspects
[0279] The following is a non-exhaustive list of isolated aspects
of the exemplary cartridge described above which may be claimed.
These aspects are described with reference to FIGS. 11 to 15. The
inclusion of this section does not preclude there being further
aspects of the exemplary cartridge described above which may also
be claimed.
[0280] 3.1 Valves for Minimising Dead Volume
[0281] An advantageous arrangement for a valve in a fluidic
cartridge will now be described, which may form an isolated
inventive aspect.
[0282] Hence, in one aspect, there is provided a valve for a
fluidic cartridge, the valve comprising: [0283] a valve cavity
having first and second openings connected to first and second
passageways, respectively; and [0284] a flexible membrane movable
between a closed position, in which the flexible membrane seals
against the first and second openings to prevent fluid flow between
the first and second passageways, and an open position, in which
the flexible membrane is spaced apart from the first and second
openings to permit fluid to flow between the first and second
passageways; [0285] wherein the a valve cavity comprises a roof and
a floor, the floor comprising said first and second openings; and
further comprising: [0286] an abutment between the flexible
membrane and the roof of the valve cavity, such that the abutment
restricts movement of the membrane in its open position.
[0287] Preferably the abutment is provided on the flexible
membrane, and comprises one or more of a protrusion, a cage, a lip
or a cross structure.
[0288] It is sometimes advantageous to limit the extent to which
the flexible membrane in a valve described herein is able to travel
in its open position. That is, it is desirable to minimise the
distance which the valve membrane moves to its open, and thus
minimise the distance it must travel to close. By minimising this
distance, the dead volume within the valve cavity is reduced,
improving the reactivity of the valve.
[0289] Hence, as shown in more detail in FIG. 11, preferred
embodiments of a valve 300 further comprise an abutment 302. The
abutment of the illustrated example is a cross structure, but in
different embodiments may be a protrusion, cage, lip or similar,
attached to the upper surface of the flexible membrane 304 so as to
contact the roof 306 of the valve cavity and thus limit movement of
the membrane in its open position.
[0290] It should be appreciated that the channels and openings of
the valve are not shown in FIG. 11.
[0291] The abutment is particularly advantageous when filing the
amplification chambers of the exemplary cartridge, because it
reduces the dead-volume in the valve cavity and thus limits the
distance between the bottom surface of the flexible membrane and
the openings in the valve cavity, thereby permitting a more precise
volume of fluid to be metered into the amplification chambers.
[0292] 3.2 Crack Pressure in Valves
[0293] An advantageous arrangement for a valve in a fluidic
cartridge will now be described, which may form an isolated
inventive aspect.
[0294] Hence, in one aspect, there is provided a valve for a
fluidic cartridge, the valve comprising: [0295] a valve cavity
having first and second openings connected to first and second
passageways, respectively; [0296] a flexible membrane within the
valve cavity movable between a closed position, in which the
flexible membrane seals against the first and second openings to
prevent fluid flow between the first and second passageways, and an
open position, in which the flexible membrane is spaced apart from
the first and second openings to permit fluid to flow between the
first and second passageways; wherein [0297] the valve is
configured such that a pressure required in the first passageway to
move the flexible membrane from the closed position to the open
position is higher than a pressure required in the second
passageway to move the flexible membrane from the closed position
to the open position.
[0298] It will be appreciated that within the valve cavity there is
a portion (known as the valve chamber) between the flexible
membrane and the floor. There is also a portion within the valve
cavity on the other side of the flexible membrane to the valve
chamber. This portion will have a volume. The pressure within that
volume may be changed by applying a positive or gauge pressure to
the volume via an actuation channel, for example. The actuation
channel may be connected to a source of positive or gauge pressure
via a pneumatic interface, for example. The pressure within the
volume is known as the actuation pressure. This operation is
described in more detail above.
[0299] In a preferred arrangement, the first and second openings
may be arranged such that fluid in the first passageway acts on the
flexible membrane only over a relatively small cross-sectional area
whereas fluid in the second passageway acts on the flexible
membrane over a larger cross-sectional area, preferably
substantially the whole membrane.
[0300] The effect of this is that the valve is able to withstand a
much greater pressure in the first passageway that in the second
passageway.
[0301] Preferably the valve cavity has a floor comprising the first
and second openings and one or more walls between which the
flexible membrane extends; and wherein the second opening is
coupled to a recess in the floor between the opening and the
flexible membrane, the recess having a larger cross-sectional area
than the opening.
[0302] Preferably the first opening is located centrally within the
floor and the recess extends around the first opening, such that
the second opening is located between the first opening and a wall
of the valve cavity. In a particularly preferred arrangement, the
valve cavity has a circular cross section, and the recess is an
annular recess which surrounds the first opening.
[0303] Preferably the opening of the second fluid passageway is
located adjacent the perimeter of the valve chamber. Preferably the
valve chamber has a diameter of between 2 and 10 mm, preferably
between 3 and 7 mm and more preferably 4 and 6 mm. More preferably,
the second opening is offset by 2 mm from the first opening.
[0304] An exemplary valve is shown in FIG. 12 in its closed
position. The valve 310 may be used in place of any of the valves
of the exemplary fluidic cartridge shown above. The valve comprises
a valve cavity 312 having a flexible membrane 314 overlying a
cavity floor 316 in which first 318 and second 320 apertures are
provided, leading to first 322 and second 324 fluid passageways,
respectively.
[0305] The cavity 312 is formed from a void in a first polymer
layer (preferably the fluidic layer 114 of the exemplary cartridge)
together with a second polymer layer (preferably the second fluidic
layer 115 of the exemplary cartridge).
[0306] The flexible membrane 314 is shown lying across the floor
316 of the cavity such that the valve is shown in its closed
position. The valve is movable from this position to an open
position (where it is spaced from the floor 316 and the apertures
322, 324 to form a valve chamber), as described herein.
[0307] The first opening 318 of the valve is centrally located
within the perimeter of the void formed in the first polymer layer,
and is therefore centrally located in the valve cavity 312. The
second opening 324 of the valve is offset from the first opening
322. The second opening is coupled to an annular recess 326 in the
floor, and thus the cross-sectional area over which the fluid in
the second passageway 324 acts on the flexible membrane 314 is much
larger than the cross-sectional area over which the fluid in the
first passageway 322 acts on the flexible membrane.
[0308] The pressure of a fluid in the first passageway acts on the
flexible membrane only over a relatively small cross-sectional area
of the flexible membrane. Thus, because the pressure of a fluid in
the valve cavity on the other side of the flexible membrane acts
over the whole membrane, it may be lower without allowing the
membrane to move to its open position.
[0309] In contrast, the pressure of a fluid in the second
passageway acts on the flexible membrane over a relatively large
cross-sectional area of the flexible membrane. Since the respective
cross-sectional areas are closer, so too is the pressure in the
second passageway which the flexible membrane is able to withstand
vis-a-vis the pressure in the valve cavity.
[0310] Preferably, the respective cross-sectional areas of the
openings of the fluid passageways allows the membrane to resist
pressures around 2.5 times the actuation pressure on the first,
central, fluid passageway, but only pressures equal to the
actuation pressure (i.e. the pressure in the valve cavity) on the
opening of the second, offset, fluid passageway.
[0311] 3.3 Entry Port Design
[0312] An advantageous arrangement for an entry port on a fluidic
cartridge will now be described, which may form an isolated
inventive aspect.
[0313] Hence, in one aspect, there is provided a fluidic cartridge
for processing a liquid sample, the cartridge having a sample
mixing chamber comprising: [0314] a sample inlet aperture for
introducing a liquid sample into the sample mixing chamber; [0315]
a cage surrounding the inlet aperture and extending into the sample
mixing chamber, the cage further comprising one or more protrusions
extending radially inwardly to abut against a sample delivery
device introduced through the sample inlet.
[0316] The body of the cage may be formed from one or more elongate
bars, or one or more solid walls, depending from the roof of the
sample mixing chamber. If solid walls are provided, there is
preferably an aperture in the lower portion of the walls through
which a liquid sample introduced by the sample delivery device can
pass. Preferably the bars or wall forming the body are tapered to
conform to the nib of a conventional sample delivery device
introduced through the sample inlet.
[0317] Solid walls have the additional advantage that they provide
a barrier to prevent fluid introduced into the mixing chamber from
escaping out of the inlet aperture, which is particularly useful if
the cartridge is turned upside-down during use.
[0318] If the cage is formed from solid walls, the protrusion may
be a ledge extending inwardly from the walls leaving an aperture.
Preferably the protrusion extending from the sides of the inlet
aperture is positioned above the floor of the sample mixing
chamber; more preferably above a liquid fill level of the sample
mixing chamber. This prevents liquid sample from being sucked back
into the sample delivery device once introduced into the mixing
chamber.
[0319] Preferably a vent is provided in the sample mixing chamber
to allow air to escape from the chamber during the introduction of
the sample. This is particularly useful when the inlet aperture is
sealed by the sample delivery device.
[0320] Preferably a guide channel is provided within the sample
mixing chamber (a portion of which is preferably directly
underneath the inlet aperture) to direct the sample introduced by a
sample delivery device into a visual indicator region. An exemplary
visual indicator region is described above in connection with the
exemplary cartridge.
[0321] Preferably a change in refractive index of the visual
indicator region described herein identifies when a sample has been
introduced. The visual indicator region may comprise a narrow fluid
passageway, which becomes filled by the fluid sample via capillary
action. The filling of the narrow fluid passageway changes the
refractive index of the visual indicator region and a colour change
identifies when a sample has been introduced.
[0322] A preferred embodiment of this aspect will now be described
with reference to the exemplary fluidic cartridge. The housing 111
(see FIG. 4) comprises a sample inlet aperture 126 through which a
sample may be introduced into the sample mixing chamber 10 of the
cartridge 100 using a pipette, for example. As shown in more detail
in FIG. 13a, the sample mixing chamber 10 is formed from the
pneumatic layer 114, which has a roof adjacent the housing 111 in
the region of the inlet aperture, and a corresponding inlet
aperture through which a sample may be introduced into the sample
mixing chamber 10.
[0323] The roof of the mixing chamber 10 comprises a cage structure
formed by walls 330 surrounding the inlet aperture 126 which extend
into the sample mixing chamber 10 from the roof, and a ledge 332
extending radially inwardly from the walls 330. The shape of the
cage structure allows a sample delivery device, such as a pipette,
to be located in the correct position in the sample mixing chamber
10, and the ledge 332 prevents the pipette contacting the surfaces
of the sample mixing chamber 10, thereby reducing the risk of
contamination. The walls 330 can be tapered to further increase the
engagement with the pipette.
[0324] Once the sample delivery device is located through the
aperture, the user can dispense the sample. The ledge 332 is
positioned above a nominal liquid fill level (not shown) of the
sample mixing chamber so as to prevent the user from accidentally
sucking the sample back up after dispensing it into the
chamber.
[0325] A vent 334 into the chamber is provided to allow air to
escape in the event that the inlet aperture is sealed by the sample
delivery device.
[0326] A guide 336 is provided within the sample mixing chamber 10,
a portion of which is directly underneath the inlet aperture 126 to
direct the sample introduced by a sample delivery device into a
visual indicator region 338. An exemplary visual indicator region
is described above in connection with the exemplary cartridge.
[0327] 3.4 Thermal Isolation Pockets
[0328] An advantageous arrangement for thermal isolation pockets
for a nucleic acid amplification chamber on a fluidic cartridge
will now be described, which may form an isolated inventive
aspect.
[0329] In nucleic acid amplification and detection, it is
preferable to apply heat evenly throughout the liquid sample.
Whilst it is possible to do this without difficulty in a laboratory
by placing heat sources equidistantly around the sample, it is much
harder to achieve in a cartridge.
[0330] Hence, in one aspect, there is provided a fluidic cartridge
for performing nucleic acid amplification on a liquid sample, the
cartridge comprising at least one sample processing chamber and a
thermally insulating region adjacent the chamber to prevent heat
loss from the chamber through the thermally insulating region.
Preferably the at least one sample processing chamber is one or
both of a nucleic acid amplification chamber and a nucleic acid
detection chamber (hence forth `processing chamber`).
[0331] Preferably the nucleic acid processing chamber is adjacent a
surface (preferably a bottom surface) of the cartridge for
accepting heat from an external source, the chamber situated
between the thermally insulating region and the surface such that
heat passing from the external source through the surface and
thence the chamber is not lost out of the other side of the chamber
owing to the presence of the thermally insulating region. This
arrangement is found to make the change in temperature inside the
chamber (for instance when turning the heat source on and off) as
fast as possible, which is beneficial for performing rapid PCR, for
example.
[0332] This is particularly advantageous because a single heat
source may be placed adjacent the cartridge to supply heat for the
amplification process from one side (the heated side), and yet the
sample within the cartridge will be heated substantially and the
amount of heat lost through the unheated side minimised as far as
possible.
[0333] Preferably the cartridge is comprised of at least a fluidic
layer and a pneumatic layer in contacting arrangement. The nucleic
acid processing chamber may be formed in the fluidic layer and the
thermally insulating region may be formed in the pneumatic layer.
Preferably the fluidic cartridge further comprises a fluidic foil
underneath the fluidic layer, the foil forming the aforementioned
surface for accepting heat. The use of a thin foil maximises the
heat transfer from the external source. The material of the foil
may be chosen to optimise the heat transfer. For instance, a metal
foil may be used, but it is preferred that a polyethylene
terephthalate/polypropylene composite is used due to the advantages
in ease of manufacture of the cartridge, together with material
strength and acceptable heat transfer properties.
[0334] Preferably the thermally insulating region is formed from
one or more sealed thermal isolation pockets formed in the
pneumatic layer and sealed by a pneumatic foil. The pockets may be
filled with gas such as air or may be evacuated during the
manufacturing process such that they provide a vacuum.
[0335] A preferred embodiment of this aspect will now be described
with reference to the exemplary fluidic cartridge. As shown in FIG.
3, the exemplary cartridge 100 comprises, from top to bottom, a
housing 111, a blister sub-assembly 112, a pneumatic foil 113, a
pneumatic layer 114, a fluid layer 115 and a fluidic foil 116.
[0336] Referring to FIGS. 6A and 6B, which shows the pneumatic
layer, six thermally insulating regions 140a-b, 141a-d are
provided. The insulating regions 140a-b are located adjacent two
corresponding amplification chambers formed in the fluidic layer
115, whilst insulating regions 141a-d are located adjacent four
corresponding detection chambers formed in the fluidic layer 115,
when the cartridge is assembled. As shown, the insulating regions
140a-b consist of a plurality of thermal isolation pockets, whereas
insulating regions 141a-d each consist of a single pocket.
[0337] During nucleic acid amplification and detection,
thermocycling of the amplification and detection chambers takes
place. The chambers in the fluidic layer may be heated by applying
heat to the bottom of the cartridge 100, adjacent the fluidic layer
115. The thermal isolation pockets retain the heat within the
cartridge, minimising heat loss from the fluidic layer 115 into the
pneumatic layer 114. The thermal isolation pockets also eliminate
the need for heating of the fluidics cartridge from both the top
and bottom surfaces e.g. heating both the fluidics layer and the
pneumatic layer, simplifying the overall design of the cartridge
and reader.
[0338] The thermal isolation pocket may comprise one large pocket
or multiple smaller pockets. The advantage of using multiple
smaller pockets is that the risk of convection currents being set
up is reduced, thus providing maximal thermal insulation.
[0339] 3.5 Capture Column
[0340] An advantageous arrangement for a filtering device in a
fluidic cartridge (preferably a `capture column`) will now be
described, which may form an isolated inventive aspect.
[0341] Hence, in one aspect, there is provided a fluidic cartridge
comprising a channel through which a liquid sample may pass, the
channel having a filter for capturing biological components and
further comprising: [0342] an upstream portion and a downstream
portion; and [0343] a capture portion between the upstream and
downstream portions in which the filter is arranged; wherein:
[0344] the diameter of the capture portion is greater than the
diameter of the upstream and downstream portions.
[0345] Preferably the capture portion is a chamber within the
channel, the chamber having an inlet surface having an opening
coupled to the upstream portion of the channel and an outlet
surface having an opening coupled to the downstream portion of the
channel.
[0346] Preferably the fluidic cartridge comprises at least two
polymer layers, wherein the upstream portion and an upstream part
of the capture portion of the channel are formed in a first polymer
layer and the downstream portion and a downstream part of the
capture portion of the channel are formed in a second polymer
layer; and wherein the filter is clamped between the first and
second polymer layers.
[0347] Preferably the inlet surface of the chamber comprises
distribution conduits leading radially outwardly from the opening
so as to direct a liquid sample passing through the opening in the
inlet surface radially outwardly.
[0348] Preferably the outlet surface of the chamber comprises
distribution conduits leading radially inwardly toward the opening
so as to direct a liquid sample which has passed through the filter
radially Inwardly toward the opening in the outlet surface.
[0349] A preferred embodiment of this aspect will now be described
with reference to the exemplary fluidic cartridge. In the exemplary
cartridge described herein, a capture column 24 is provided along
the main channel (see FIG. 1). As shown in FIGS. 14a and 14b, the
capture column 24 has filter 340 which binds DNA from lysed
material before releasing it during elution. As shown in FIG. 14a,
capture column 24 comprises an inlet channel 342 leading into a
capture chamber 344 at an upstream end 346, and an outlet channel
350 leading from capture chamber 344 at a downstream end 348.
[0350] A filter 340 is provided in chamber 344, perpendicular to
the direction of flow of fluid through the main channel, such that
fluid must pass through filter 340 when passing from the upstream
end of the main channel 342 to the downstream end 350 of the main
channel.
[0351] Referring now to FIG. 14b, the inlet and outlet walls (only
one is shown) of the chamber comprise distribution conduits 352
configured to direct fluid radially outwardly into the chamber 344
as it enters the chamber, and radially inwardly toward the exit
aperture after it has passed through the filter 340.
[0352] 3.6 Waste Chamber
[0353] An advantageous arrangement for waste chamber in a fluidic
cartridge will now be described, which may form an isolated
inventive aspect.
[0354] Hence, in one aspect, there is provided a fluidic cartridge
comprising a channel through which a liquid sample may pass and a
waste chamber for receiving fluid from the channel, the waste
chamber comprising: [0355] a pipe, coupled to the channel,
extending from a bottom surface of the waste chamber and having an
opening elevated above the bottom surface to pass fluid from the
channel into the chamber; and [0356] a vent within the waste
chamber configured to vent the waste chamber to atmosphere.
[0357] Preferably the vent comprises a second pipe, coupled to a
vent channel within the cartridge, extending from the bottom
surface of the waste chamber and having an opening elevated above
the bottom surface. Preferably the vent passageway comprises at
least one Anderson impactor.
[0358] Preferably at least one absorbent pad is provided within the
waste chamber.
[0359] A preferred embodiment of this aspect will now be described
with reference to the exemplary fluidic cartridge. In the exemplary
cartridge described herein, a waste chamber is provided for
collecting and storing waste fluid which is produced during washing
etc. Waste chamber 10 is shown in more detail in FIGS. 15a and 15b.
Waste chamber 38 comprises a pipe 360, extending substantially
vertically from a bottom surface 362 of waste chamber 38. The pipe
38 defines a channel having a first end 364 connected to the bottom
surface of the waste chamber 38 and fluidly connected to the main
channel 16. A second end 366 of fluid pipe 360 is disposed within
waste chamber 38, and has an opening through which fluid can flow
into the waste chamber.
[0360] Preferably, pipe 360 is substantially vertical, and
perpendicular to the bottom surface of the waste chamber 38. The
opening at the second end of pipe 360 is located near the top of
the waste chamber 38 as shown in FIG. 15b. By providing the first
opening near the top of the waste chamber, the risk of leakage is
minimised should the cartridge be turned upside down.
[0361] Absorbent pads 368 are also provided in the waste chamber.
Preferably, the upper surface of absorbent pads 368 should also be
near the top of waste chamber 38, even more preferably, the top of
absorbent pads 368 should be substantially level with the opening
at the second end 366.
[0362] In the exemplary cartridge described herein, a second
opening 370 is provided in waste chamber 38 as shown in FIG. 15b
The second opening 370 is configured to vent main channel 16 via
waste chamber 28 to atmospheric pressure. This avoids putting a
back pressure along the main channel as the waste channel fills
with fluid. Preferably, the second opening 370 is provided at the
end of a second pipe 372 protruding from the bottom surface of
waste chamber 38. The second opening 370 may be fluidly connected
to a vent passageway (not shown) which has an opening outside of
the cartridge housing to allow the waste chamber to remain at
atmospheric pressure. However, venting the waste chamber outside
the cartridge carries a small risk of aerosol contamination. To
reduce this, the vent path has impact traps and vents under the
cartridge cover.
[0363] The skilled person will be capable of modifying the
exemplary cartridge to implement the inventive aspects described
herein in various ways depending on circumstances. It is intended
that the scope of the present invention is defined by the following
claims.
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